NO783976L - PROCEDURE FOR PREPARING A CLEANED CAPSULATED POLYSACCHARIDE OF STREPTOCOCCUS PNEUMONIAE - Google Patents

Description

Process for producing a purified capsular polysaccharide of Streptococcus pneumoniae

The present invention relates to a multivalent vaccine for bacterial pneumonia. The vaccine comprises a number of capsular polysaccharides of different types of Streptococcus pneuraoniae. Each capsular polysaccharide is produced by growing the organism that produces the capsular polysaccharide under controlled pH conditions and supplementing the glucose concentration during fermentation. pH is maintained at from approx. 6.2 to approx. 7.0 by adding the required amount of an alkaline reagent such as NaHC03>-Na2C03, NaOH, KHCO^ ^ 2C0^, KOH, Mg(OH)2, Ca(OH)2 or Al(OH)3- Buffer solutions can also be used to regulate pH, but are less desirable because of the relatively large volumes of buffer solution required. A lyophilized culture is conveniently used as the source of the organism. After fermentation, the polysaccharide is obtained by a two-stage alcoholic precipitation followed by the removal of proteins and nucleic acids. In the first alcohol precipitation, a pre-determined amount of water-miscible alcohol is added to a liquid material containing the polysaccharide to precipitate contaminants while keeping the polysaccharide in solution. After removal of the trapped contaminants, another predetermined quantity of a water-miscible alcohol is added. The amount selected for this precipitation is sufficient to precipitate all or substantially all of the capsular polysaccharide without precipitating a significant amount of other material. The liquid material containing the polysaccharide may be a fermentation broth in which the desired type of Streptococcus pneuraoniae has been grown. Although any water-miscible alcohol can be used in the precipitation, preferably an alcohol having one or two carbon atoms is used.

The precipitated polysaccharide is resuspended in liquid medium and processed to remove proteinaceous material and nucleic acids. This can be done by digesting the liquid medium containing the capsular polysaccharide with an enzyme such as trypsin or nucleases such as ribonuclease or deoxyribonuclease. Alternatively'

can the liquid material containing the capsular poly-

saccharide is treated with a cationic surfactant such as cetrimonium bromide (N,N,N-trimethyl-1-hexadecanaminium bromide). If necessary, prot einfra gment is , e.g. peptides and nucleic acid fragments are removed by appropriate treatment, e.g. by dialysis. The highly purified capsular saccharide of the desired type of Streptococcus pneuraoniae is then recovered by precipitation with a water-miscible alcohol. An alcohol with 2 or 3 carbon atoms is preferably used.

The amount of alcohol or cetrimonium bromide required in the preceding methods is determined by a preliminary investigation.

2 ml portions, as many as there are fractions to sub-

is sought, of the entire fermentation liquid (or other aqueous solvent

ning) are introduced into l6 x 25 mm disposable culture glasses. The fractions to be examined are usually taken at 5% intervals over a range 10-15% exceeding the expected precipitation concentration previously found for a particular type. If e.g. the expected interval is 40-45%, the following fractions should under-

wanted: 0-35%, 30-35%, 35-40%, 4o-45%, 45-50% and 5o-55%.

Alcohol is added while stirring with a test tube stirrer

to the lower level of each area. Stirring is necessary to

avoid local overconcentration which will lead to premature shedding. All test tubes are centrifuged on a bench-top clinical centrifuge (7-10,000 r/min) for 10 minutes.

The supernatant liquid is decanted into a 16 x 125 ml test tube, and alcohol is added, again with stirring, up to the upper limit for each fraction. All pellets except the first

(representing material in the O-low limit of the range)

thrown away.

The tubes are centrifuged again for 10 minutes, and the supernatant liquid is discarded. The pellets in each test tube now represent material fields in the individual alcohol fractions. Each

pellet is dispersed (polysaccharide extracted) in 2 ml

"Dulbecco's PBS" (lx). The resulting suspension is clarified for insoluble material with a "Swinnex-25" filter ("Millipore")

placed on a disposable syringe. A part of the filtrate from each fraction is diluted 1:2 and 1:4 with "PBS". The fractions are now ready for examination.

Antisera as supplied, first dilute 1:10 with "PBS" (lx),

and a portion (about 1.2 ml) of this is further diluted 1:6.7; sufficient to judge all factions in a particular investigation. In general, these dilutions work. Occasionally, however, a batch of antisera has been found to be less "strong", and more concentrated dilutions must be prepared. This cannot be predicted until experience with a particular batch is gained.

0.2 ml of diluted antiserum is introduced into a series of 10 x 75 ml disposable culture dishes, three for each fraction interval.

In each test tube containing antiserum introduce 10 µl

(for ethanol studies) of the fractions to be combined at the three concentrations (undiluted, 1:2, 1:4)» 5 M-l is used for isopropanol studies. The test tubes are stirred and left for 15 minutes to 1 hour. For convenience, they are usually arranged in the following networks.

A positive shedding reaction is indicated by a clear flatness or a real shedding. The desired polysaccharide can be cleaved over two neighboring fractions, or over three fractions where the middle one gives a clearer precipitin reaction. Both of these are positive samples, and the process alcohol or cetavlon fractionation range is selected on the basis of these results.

'There is an optimum concentration for the precipitin reaction,

and this is the reason for the three different dilutions. An excess of antibody may lead to no fallout, while an excess of antigen often indicates positive results over a large area, sometimes with a weak reaction between two stronger ones. In this case, the weak reaction is in reality the strongest positive, and this can be seen in the more diluted (in antigen)

sew st em .

All Streptococcus pneuraoniae type designations used here are US type designations as opposed to Danish type designations. ATCC collection numbers for the different types are as follows:

A vaccine can be prepared by incorporating a purified capsular polysaccharide in a suitable physiologically acceptable

medium such as saline, water for injection or phosphate buffered saline. Multivalent vaccines can be prepared by incorporating two or more of the purified capsular polysaccharides in a suitable physiologically acceptable medium.

A detailed description of the fermentation process to form pneuraococcal polysaccharide follows.

A lyophilized "L" tube culture is suspended in 1 ml of "Difco

Heart Infusion Broth" (HIB broth), and 0.2 ml is spread on a rabbit blood agar plate (HIB). After about 18 hours of incubation at 37"c, the growth on the plate is resuspended in 5 ml of HIB broth, and 1 ml of the suspension is transferred into 100 ml of rabbit blood-liquid medium and incubated as a stationary culture for 18 hours at 37°C. After incubation, the culture is examined microscopically and streaked on YED

and HIB plates to check purity. The 100 ml culture is stored in a refrigerator at 4°C for a maximum of 1 week.

20 ml of the previous culture is used to inoculate

1 liter of inoculum medium in a 2-liter Erlenmeyer flask with a side arm for adding sodium bicarbonate. The flask is incubated stationary at 37°C until the specifications for the particular type are satisfied. The pH of the fermentation is regulated (6.2-7.0) by adding 12% sodium bicarbonate using phenol-

red in the medium as an indicator. A final sample is examined microscopically, and an agglutination test is performed to check purity and type. If the obtained culture is not used immediately,

it can be stored for 3-4 hours at 4°C. However, it is preferred to use the culture immediately in the following step.

1 liter from the previous step is used to inoculate

a 14-liter fermenter containing 9 l of kimraedium. The batch is inoculated at 37°C under gentle stirring (100 r/min) without air

current. The course of the fermentation is followed by o.d., pH and glucose determinations. When a specific OD is reached, the batch is examined microscopically, and an agglutination reaction is performed, and a sample applied to YED and HIB plates is examined for purity. The pH is adjusted during fermentation (6.2-7.0) using 10% sodium hydroxide. If desired, a portion of this culture can be used to inoculate a larger fermenter using the preceding method.

When the batch is finished, the culture is applied to YED and HIB plates, examined for purity by Gram's stain, and identified with respect to type by the agglutination reaction. The fermentation liquid is then inactivated by harvesting in pre-liquefied phenol (89%) to a final concentration of 1%. After 2 hours in phenol, the fermentation liquid is released to extract the product. A sample is taken and applied to the HIB to check for viability.

The following culture media are used in the fermentation processes.

1. YED plates

Yeast extract straight 10 g/l

Dextrose 10 g/l

Agar 20 g/l

2. HIB broth "Heart Infusion Broth" 25 g/l

3. Agar rabbit blood medium (HIB)

"Heart Infusion Broth" 25 g/l

Agar 20 g/l

The above is autoclaved for 20 minutes and cooled to 50°C, and 10% defibrinated rabbit blood is added.

4. Canine blood fluid medium

Same as above except that the agar is looped.

5. Inoculum medium - 2 liters "Hysoy" - Humko Sheffield 20 g

"Amberex 1003" 10 g

NaCl 5 g

k2h<p>o^2.5 g

Phenol-red 10 g

Distilled H20 to 900 ml

The pH is set to 7>2, and the medium is autoclaved for 25 minutes at 121°C.

Glucose 25 g

The glucose is autoclaved separately in 100 ml of distilled H2O for 20 minutes and added aseptically to the above ingredients.

6. 14 liters of germination medium for type 20 and production medium for all other types

"Hysoy" - Humko Sheffield 180 g

"Amberex 1003" 90 g

NaCl 45 g K„HP0, '22.5 g

2 4

Phenol-red 90 rag

"UCON LB 625" 8% solution

(pre-sterilized for 1 hour) 40 ml

Distilled H20 to 8 liters

The pH is adjusted to 7.2 before sterilization. The above components are autoclaved together for 90 minutes at 121°C.

Glucose 225 Q

Distilled water to 1 1 litres

The glucose is autoclaved separately for 25 minutes at 121°C and added aseptically to the above ingredients.

7- Macroscopically prepared glass agglutination identification sample a. A loopful of specific Pneumococcus antiserum

(specific type) is placed on a preparation glass.

b. A loopful of Streptococcus pneuraoniae (specific type)

from the kit is transferred to the drop of antiserum and mixed.

c. A positive sample is obtained if an agglutination of the cells occurs with specific antisera.

8. Procedure for demonstrating viability

A 0.5 ml sample of the broth to be checked for viable organisms is spread on "Heart Infusion Blood" agar. The plates are incubated at 37°C for 48 hours. No visible signs of growth after 48 hours represent negative viability.

Pneumococcal polysaccharide type 19 is indicated in the literature

to consist of L-rharanose, D-glucose, 2-acetamido-2-deoxy-D-mannose and phosphate in an approximate molar ratio of 2:1:1:1 (Miyazaki et al., Carbohydrate Research, Vol. l6, p 153-159, 1971). Based on this publication, the theoretical amount of L-rhamnose in type 19 is 43%, and the theoretical amount of D-glucose is 21.9% - Type 19 saccharide product obtained according to the present invention, however, contains approx. 15-20% by weight of L -rhamnose and approx.

5.5-8% by weight D-glucose. It has also been shown to contain approx.

8-11% by weight 2-acetamido-2-deoxy-D-mannose (N-acetyl-mannosamine).

Pneumococcal polysaccharide type 23 is stated in the literature to consist of rharanose, glucose and galactose in a molar ratio of 5:2:2 (Heidelberger et al., Journal of Immunology, Vol. 99,

pp. 794-796, 1967). Based on these conditions, the theoretical amount of L-rhamnose is 59.5%. Larm et al., in Advances in Carbohydrate Chemistry and Biochemistry, Tipson & Horton, editors,

Acadeinic Press, 1967, p. 314, states that pneumococcal polysaccharide type 23 contains 35% L-rhamnose, 31% D-galactose and 32% D-glucose. Type 23 polysaccharide product obtained according to the present invention, however, contains rhamnose, galactose and glucose in the molar ratio 2:1:1, and contains approx. 31_38% by weight L-rhamnose, approx. 15-18% by weight D-galactose and approx. 15-18% by weight

D-glucose.

Pneumococcal polysaccharide type 25 is stated to contain galacturonic acid as its uronic acid (Heidelberger, Immunochemistry of Bacterial Polysaccharides, in Research in Immunochemistry and Immunobiology, Vol. 3, pp. 1-40, Kwapinski & Day, editors, University Park Press, Baltimore, 1973) • Type 25 polysaccharide product obtained according to the present invention contains approx. 2-4% by weight fucose, approx. 2-4% by weight mannose, approx.

15-19% by weight galactose, approx. 1.8~4% by weight D-glucose, approx. 7~9% by weight galactosamine, approx. 15-19% by weight glucosamine and approx. 5~7% by weight uronic acid.

Pneumococcal polysaccharide type 72 product obtained according to the present invention contains approx. 5.5~8% by weight rhamnose, approx. 3-6.5% by weight fucose, approx. 2.7~4.8 wt% mannose,

about. 17-22% by weight D-galactose, approx. 11.5-16.5% by weight galactosamine and approx. 27-32 wt% glucosamine.

The pneumococcal polysaccharide type 73 product obtained according to the present invention contains approx. 1.8-3.5% by weight fucose, approx. 2-5% by weight mannose, approx. 14~19% by weight D-galactose,

about. 12-15% by weight of galactosamine and approx. 29~34% by weight glucosamine.

The above carbohydrate analyzes for the types 19, 23, 25, 72 and 73 polysaccharide products according to the present invention have been determined by gas chromatography.

The following examples further illustrate the present invention, but do not limit it.

Example 1

Pneumococcal polysaccharide type 1

A culture of Streptococcus pneuraoniae type 1 is grown on blood agar for 16 hours at 37°C. The cells are collected and placed in 250 ml of blood broth for 16 hours at 37°C. A 5-10 ral sample of the blood broth growth is used to inoculate a 2 liter flask containing meat extract nutrient medium. The culture is grown for 16 hours at 37°C with the addition of sodium bicarbonate at 2 hour intervals to neutralize the medium. A 14 liter flask containing meat extract nutrient medium is inoculated with the contents of the 2 liter flask and grown for 16 hours at 37°C with the addition of sodium bicarbonate at 2 hour intervals to neutralize the medium. Phenol is then added to the medium to a 1% by volume concentration to kill the bacteria.

An ethanol assay is performed on a sample of the phenolic whole broth to determine the ethanol range for polysaccharide sweep. The range for this rate is from 38 to 52%.

While stirring and at room temperature, 3>68 1 of ethanol is slowly added to 6 1 of the phenol-treated whole broth, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15-000 r/min in a 10 cm Sharples centrifuge. The solids are discarded.

While stirring, a further 2.82 1 of ethanol is slowly added to the supernatant liquid, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15-000 rpm in a 10 cm Sharples centrifuge to obtain a clear supernatant which is discarded. The impure polysaccharide solid is suspended in 0.09 1 3% sodium acetate solution. The pH is adjusted to 6.5 with glacial acetic acid and stirred for 4 hours to ensure dissolution of the polysaccharide.

An isopropanol test is performed on a sample of the polysaccharide-containing liquid to determine the optimal isopropanol range for polysaccharide sweeping. The range for this rate is from 20 to 30%.

While stirring, 22.5 ml isopropanol is added slowly, and the resulting solution is aged for 4 hours with continuous stirring. The mixture is clarified at 15-000 r/min in a 10 cm Sharples centrifuge. Clarification is completed in a K-2 ultracentrifuge at 25,000-30,000 r/min. The solids are discarded. While stirring, a further 16 ml of isopropanol is added to the liquid above, and the resulting mixture is aged for 4 hours while stirring. The impure polysaccharide precipitate is recovered by centrifugation at 15,000 rpm in a 10 cm Sharples centrifuge, and the supernatant liquid is discarded.

The impure polysaccharide is suspended in 90 ml of 3% sodium acetate. The pH is adjusted to 6.5 with glacial acetic acid and 65 mg of MgSO, 2.7 mg of ribonuclease and 2.7 mg of deoxyribonuclease are added. The solution is dissolved with stirring at 37°C for 3 hours and cooled to 0-5°C.

A cetrimonium bromide assay is performed on a sample of the fluid to determine the optimum cetavlone range for polysaccharide binding. With this rate, the range is from 33-52%. While stirring, 59 ml of a 1% aqueous cetrimonium bromide solution is added, and the resulting mixture is aged for 4 hours. The mixture is clarified in a K-2 ultracentrifuge at 25,000-30,000 r/min. The solids are discarded, and a further 96.3 ml of 1% aqueous cetrimonium bromide solution is added to the supernatant liquid, and the resulting mixture is aged for 4 hours with stirring. The impure polysaccharide precipitate is removed by centrifugation at 15-000 r/min in a 10 cm Sharples centrifuge, and the supernatant liquid is discarded.

The precipitate is dissolved in 90 ml of a 20% sodium acetate solution, and the pH is adjusted to 6.5 with glacial acetic acid. 90 ml of isopropanol is added with stirring, the mixture is stirred for 2 hours and allowed to settle for several hours until a gummy precipitate is separated at the bottom. The entire liquid above is decanted, and the precipitate is triturated in a Waring blender with approx. 40 ml of absolute ethanol until a white powder is obtained. The powder is recovered by filtration on a Nutsch (Whatman No. 2 paper) while washing with 2 x 5 ml of absolute ethanol and 2 x 5 ml of acetone. The washed powder is dried at approx. 686 mm Hg and room temperature for 12-16 hours, whereby you get approx. 0.35 g of the purified polysaccharide.

Example 2

Pneumococcal polysaccharide type 2

A culture of Streptococcus pneumonia type 2 is grown on blood agar for 16 hours at 37°C. The cells are collected and placed in 250 ml of blood broth for 16 hours at 37°C. A 5-10 ml sample of the blood broth growth is used to inoculate a 2 liter flask containing meat extract nutrient medium. The culture is grown for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. A 14-liter flask containing meat extract nutrient medium is inoculated with the contents of the 2-liter flask and grown for 16 hours at 37°C

while adding sodium bicarbonate at 2 hour intervals to neutralize the medium. Phenol is then added to the medium to a 1% by volume concentration to kill the bacteria.

An ethanol test is carried out on a sample of the phenolic

ized whole broth to determine the ethanol range of polysaccharide unit . The range for this lot is from 40 to 55%.

While stirring and at room temperature, 40 liters of ethanol are slowly added to 6 liters of the phenolized whole broth, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15-000 r/min in a 10 cm Sharples centrifuge. The solids are discarded.

While stirring, a further 3.33 liters of ethanol are added slowly to the above liquid, and the resulting mixture

aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15,000 r/min in a 10 cm Sharples centrifuge without precipitation of any significant amount of other material. The liquid material containing the polysaccharide may be a fermentation broth in which the desired type of Streptococcus pneuraoniae has been grown. Although any water-miscible alcohol can be used in the precipitation, the alcohol should preferably have one or two carbon atoms.

The precipitated polysaccharide is resuspended in liquid medium and processed to remove proteinaceous material and nucleic acids.

This can be carried out by digesting the liquid medium containing the capsular polysaccharide with an enzyme such as trypsin or nucleases such as ribonuclease or deoxyribonuclease. Alternatively, the liquid material containing the capsular polysaccharide can be treated with a cationic surfactant such as cetrimonium bromide (N,N,N-trimethyl-1-hexadecanaminium bromide). If necessary,

can protein fragments, e.g. peptides and nucleic acid fragments, are removed by appropriate treatment, e.g. by dialysis. The highly purified capsular saccharide of the desired type of Strepto coccus pneuraoniae is then recovered by precipitation with a water-miscible alcohol - Preferably the alcohol has 2 or 3 carbon atoms.

The amount of alcohol or cetrimonium bromide required

for the preceding processes, is determined by a preliminary investigation. 2 ml portions, as many as there are fractions to be examined, of whole broth (or other aqueous solution) in-

placed in 16 x 25 cm disposable culture glasses. The fractions to be examined are usually taken at 5% intervals over a range 10-15% spanning the expected precipitate concentration found previously on a centrifuge to provide a clear supernatant which is discarded. The impure polysaccharide solids are suspended in 0.6 liters of 1% sodium acetate solution, and the mixture is stirred for 4 hours to ensure dissolution of the polysaccharide.

The mixture is clarified at 15-000 r/min in a 10 cm Sharples centrifuge. Clarification is completed in a K-2 ultracentrifuge at 25,000-30,000 r/min. The solids are discarded.

The pH of the supernatant liquid is adjusted to 8.0 with 50% sodium hydroxide, 12 mg of trypsin is added, and the solution is digested at 37°C for 90 minutes. The mixture is cooled to 20-25°C,

and the pH is adjusted to 6.5 with glacial acetic acid.

The dissolved solution is cooled to 0-5°C, and 7.2 g of cetrimonium bromide are added. The cetrimonium bromide solution is aged for 4 hours at 0-5°C and centrifuged in a K-2 ultracentrifuge at 25.000-30.000 r/min. The solids are discarded.

120 g of sodium acetate is added to the liquid followed by 0.6 l of ethanol. The mixture is stirred for 2 hours, and the precipitate is removed by centrifugation in a 10 cm Sharples centrifuge at 15-000 rpm. The precipitate is triturated with 10-20 ml of 66% ethanol/water and extracted

by filtering on a Nutsch.

The impure polysaccharide is suspended in 300 ml of 3% sodium acetate solution, and the pH is adjusted to 6.5 with glacial acetic acid, and the mixture is not stirred for 4 hours to ensure dissolution of the polysaccharide.

An isopropanol test is carried out on a sample of the liquid

to determine the optimal isopropanol range for polysaccharide sweep. With this rate, the range is from 28~42% by volume. While stirring, 117 ml of isopropanol is added, and the resulting mixture is aged for 4 hours. The mixture is clarified in a 10 cm Sharples centrifuge at 15-000 r/min. Clarification is completed in a K-2 ultracentrifuge at 25,000-30,000 r/min. The solids are discarded, and a further 100 ml of isopropanol is added to the supernatant liquid, and the resulting mixture is aged for 4 hours with stirring. The impure polysaccharide precipitate is recovered by centrifugation at 15-000 rpm in a 10 cm Sharples centrifuge, and the supernatant liquid is discarded.

The precipitate is triturated in a Waring blender with approx. 40 ml of absolute ethanol until a white powder is obtained. The powder is recovered by filtration on a Nutsch (Wnatman No. 2 paper) while washing with 2x5 ml of absolute ethanol and 2x5 ml of acetone. The washed powder is dried at 686 mm Hg and room temperature for 12-16 hours, whereby approx. 4.6 g of the purified polysaccharide.

Example 3

Pneumococcal polysaccharide type 3

A culture of Streptococcus pneuraoniae type 3 is grown in blood agar for 16 hours at 37°C. The cells are collected and placed in 250 ml of blood broth for 16 hours at 37°C. A 5-10 ml sample of the blood broth growth is used to inoculate a 2 liter flask containing meat extract fermentation medium. The culture is grown for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. A 14 liter flask containing meat extract nutrient medium is inoculated with the contents of the 2 liter flask and cultured for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. Phenol is then added to the medium to a 1% by volume concentration to kill the bacteria.

An ethanol assay is performed on a sample of the phenolized whole broth to determine the ethanol range for polysaccharide precipitation. The range for this rate is from 28 to 45%-

While stirring and at room temperature, 2.33 1 of ethanol is slowly added to 6 1 of the phenolized whole broth, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15,000 r/min in a 10 cm Sharples centrifuge. The solids are discarded.

While stirring, a further 2.58 1 ethanol is added long-

as to the liquid above, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged

at 15-000 r/min in a 10 cm Sharples centrifuge. The supernatant liquid is discarded, and the impure polysaccharide solids are suspended in 1.8 1 3% sodium acetate solution using a high-shear mixer. The pH is adjusted to 6.5 with glacial acetic acid and stirred for 4 hours to ensure dissolution of the polysaccharide.

An isopropanol test is performed on a sample of the polysaccharide-containing liquid to determine the optimal isopropanol range for polysaccharide sweeping. The range for this rate is from 18 to 30% by volume.

While stirring, add 0.395 1 isopropanol slowly, and

the resulting mixture is aged for 4 hours with continuous stirring. The mixture is clarified at 15-000 r/min in a 10 cm Sharples centrifuge. Clarification is completed in a K-2 ultracentrifuge at 25,000-30,000 r/min. The solids are discarded. While stirring, a further 0.376 1 of isopropanol is slowly added to the

end liquid, and the resulting mixture is aged for 4 hours with stirring. The impure polysaccharide bottom phase 11 is recovered by centrifugation at 15-000 r/min in a 10 cm Sharples centrifuge, and the supernatant liquid is discarded.

The crude polysaccharide is suspended using a high shear mixer in 1.0 L pH 7.0, 0.05 M phosphate buffer. The pH is again adjusted to 7.0 with glacial acetic acid, and 60 mg of MgCl 2 , 0.6 mg of ribonuclease and 0.6 mg of deoxyribonuclease are added. The solution is dissolved with stirring at 37°C for 60 minutes. The pH is adjusted to 8.0 with 50% sodium hydroxide, 18 mg of trypsin is added, and the digestion

continued at 37°C for an additional 90 minutes. The mixture

cooled to 20-25°C, and pH adjusted to 6.5 with glacial acetic acid.

• The absorbed solution is diafiltered in a "Romicon

hollow filter ult rafilt rat ion membrane" until the electrical led-

ning ability of the perrnate reaches a constant value. The reservoir is kept at a constant volume with pyrogen-free water. The restrained,

containing the polysaccharide, is withdrawn from the system, and the volume is restored to 1.0 1 with system wash. 30 g of sodium acetate are added to the solution of the polysaccharide-containing liquid, and the pH is adjusted to 6.5 with glacial acetic acid. While stirring, 2.0 1 of isopropanol is added, and the resulting mixture is stirred for 15 minutes and allowed to settle for several hours until a gummy precipitate is separated at the bottom. The entire liquid above is decanted, and the precipitate is triturated in a Waring blender with approx. 40 ml absolute ethanol until a/white powder is obtained. The powder is recovered by filtration on a Nutsch (Whatman No. 2 paper) while washing with 2 x 5 ml of absolute ethanol and 2 x 5 ml of acetone. The washed powder is then dried at 686 mm Hg and room temperature for 12-16 hours, whereby approx. 2.76 g of the purified polysaccharide.

Example 4

Pneumococcal polysaccharide type 4

A culture of Streptococcus pneumoniae type 4 is grown on blood agar for 16 hours at 37°C. The cells are collected and placed in 250 ml of blood broth for 16 hours at 37°C. A 5-10 ml sample of the blood broth growth is used to inoculate a 2 liter flask containing meat extract nutrient medium. The culture is grown for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. A 14-liter flask containing meat extract nutrient medium is inoculated with the contents of the 2-liter flask and grown for 16 hours at 37°C with the addition of sodium bicarbonate at 2-hour intervals to neutralize the medium. Phenol is then added to the medium to a 1% by volume concentration to kill the bacteria.

An ethanol assay is performed on a sample of the phenolized whole broth to determine the ethanol range for polysaccharide precipitation. The range for this rate is from 68 to 80% by volume.

While stirring and at room temperature, 12.75 1 of ethanol is added slowly to 6 1 of the phenolized whole broth, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15-000 r/min in a 10 cm Sharples centrifuge. The solids are discarded.

While stirring, a further 11.25 1 ethanol is added long-

as to the liquid above, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged

at .15,000 r/min in a 10 cm Sharples centrifuge. The supernatant liquid is discarded, and the impure polysaccharide solid is suspended in 0.6 1 3% sodium acetate solution using a high-shear

mix . The pH is adjusted to 6.5 with glacial acetic acid and stirred for 4 hours to ensure dissolution of the polysaccharide.

An isopropanol test is performed on a sample of the polysaccharide-containing liquid to determine the optimal isopropanol range for polysaccharide sweeping. The range for this rate is from 42 to 55% by volume.

While stirring, 0.434 1 isopropanol is added slowly, and

the resulting mixture is aged for 4 hours with continuous stirring. The mixture is clarified at 15-000 r/min in a 10 cm Sharples centrifuge. The clarification is completed in a K-2 ultra ra centrifuge at 25,000-30,000 r/min. The solids are discarded. While stirring, a further 0.3 1 of isopropanol is added slowly to the liquid above, and the resulting mixture is aged for 4 hours while stirring.

The impure polysaccharide precipitate is recovered by centrifugation at 15-000 r/min in a 10 cm Sharples centrifuge, and the supernatant liquid is discarded.

The impure polysaccharide is suspended using a high shear mixer in 0.3 L pH 7.0, 0.05 M phosphate buffer. The pH is reset to 7.0 with glacial acetic acid and 60 mg MgCl 2 , 0.6 mg ribonuclease and 0.6 mg deoxyribonuclease are added. The solution is dissolved with stirring at 37°C for 60 minutes. The pH is adjusted to 8.0 with 50% sodium hydroxide, 18 mg of trypsin is added, and digestion is continued at 37°C for a further 90 minutes. The mixture of

cooled to 20-25°C, and pH adjusted to 6.5 with glacial acetic acid.

The dissolved solution is diafiltered in a "Romicon

hollow filter ult rafilt rat ion membrane" until the electrical conductivity of the permeate reaches a constant value. The reservoir is kept at constant volume with pyrogen-free water. The retained,

containing the polysaccharide, is drained from the system, and the volume is restored to 0.3 L with system wash. 9 g of sodium acetate are added to the solution of the polysaccharide-containing liquid, and the pH is adjusted to 6.5 with glacial acetic acid.

While stirring, 2.0 1 isopropanol is added, and the resulting mixture

ing is stirred for 15 minutes and allowed to settle for several hours until a rubbery precipitate has separated on the bottom. The entire liquid above is decanted, and the precipitate is triturated in a Waring blender with approx. 40 ml of absolute ethanol until a white

powder is available. The powder is recovered by filtration on a Nutsch (Whatman No. 2 paper) followed by washing with 2 x 5 ml of absolute ethanol and 2 x 5 ml of acetone. The washed powder is then dried at 656 mm Hg and room temperature for 12-16 hours, whereby approx. 1.32 g of the purified polysaccharide.

Example 5

Pneumococcal polysaccharide type 6

A culture of Streptococcus pneumoniae type 6 is grown on

blood agar for 16 hours at 37°C. The cells are collected and placed in 250 ml of blood broth for 16 hours at 37°C. A 5-10 ml portion of the blood broth growth is used to inoculate a 2 liter flask containing meat extract nutrient medium. The culture is grown for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. A 14-liter flask containing meat extract nutrient medium is inoculated with the contents of the 2-liter flask and grown for 16 hours at 37°C

while adding sodium bicarbonate at 2 hour intervals to neutralize the medium. Phenol is then added to the medium to a 1% by volume concentration to kill the bacteria.

An ethanol assay was performed on a sample of the phenolized whole broth to determine the ethanol range for polysaccharide sweep. The range for this rate was from 47 to 60% by volume.

While stirring and at room temperature, 5.32 1 of ethanol is slowly added to 6 1 of the phenolized whole broth, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15-000 r/min in a 10 cm Sharples centrifuge. The solids are discarded.

While stirring, a further 3.68 1 of ethanol is slowly added to the above mixture, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15-000 r/min in a 10 cm Sharples centrifuge. The supernatant liquid is discarded, and the impure polysaccharide solids are suspended'

in 0.6 1 3% sodium acetate solution using a high-shear mixer. The pH is adjusted to 6.5 with glacial acetic acid and stirred for 4 hours to ensure dissolution of the polysaccharide.

An isopropanol assay is performed on a sample of the polysaccharide containing liquid to determine the optimal isopropanol range for polysaccharide sweeping. The range for this rate was from 35 to 47% by volume.

While stirring, 0.323 1 of isopropanol is added slowly, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is clarified at 15-000 r/min in a 10 cm Sharples centrifuge. The clarification is completed in a K-2 ultracentrifuge at 25,000-30,000 r/min. The solids are discarded. While stirring, a further 0.21 1 of isopropanol is added slowly to the liquid above, and the resulting mixture is aged for 4 hours while stirring. The impure polysaccharide precipitate is recovered by centrifugation at 15-000 rpm in a .10 cm Sharples centrifuge, and the supernatant liquid is discarded.

The impure polysaccharide is suspended using a high shear mixer in 0.3 L pH 7.0, 0.05 M phosphate buffer. The pH is adjusted again to 7.0 with glacial acetic acid and 60 mg of MgCl^, 0.6 mg of ribonuclease and 0.6 mg of deoxyribonuclease are added. The solution is dissolved with stirring at 37°C for 60 minutes. The pH is adjusted to 8.0 with 50% sodium hydroxide, 18 mg of trypsin is added, and digestion is continued at 37°C for a further 90 minutes. The mixture is cooled to 20-25°C, and the pH is adjusted to 6.5 with glacial acetic acid.

The trapped solution is diafiltered in a "Romicon hollow filter ult rafilt rat ion membrane" until the electrical conductivity of the permeate reaches a constant value. The reservoir is kept at a constant volume with pyrogen-free water. The restrained,

containing the polysaccharide, is drained from the system, and the volume is reset to 0.3 1 with system washer.

9 g of sodium acetate are added to the solution of the polysaccharide-containing liquid, and the pH is adjusted to 6.5 with glacial acetic acid. While stirring, 0.6 1 of isopropanol is added, and the resulting mixture is stirred for 15 minutes and allowed to settle for several hours until a gummy precipitate has separated at the bottom. The entire liquid above is decanted, and the precipitate is triturated in a Waring blender with approx. 40 ml absolute^ ethanol until a white

powder is available. The powder is recovered by filtration on a Nutsch (Whatman No. 2 paper) while washing with 2 x 5 ml of absolute ethanol and 2 x 5 ml of acetone. The washed powder is then dried at 686 mm Hg and room temperature for 12-16 hours, whereby approx. 4.08 g of the purified polysaccharide.

Example 6

Pneumococcal polysaccharide type 8

A culture of Streptococcus pneuraoniae type 8 is grown on blood agar for 16 hours at 37°C. The cells are collected and placed in 250 ml of blood broth for 16 hours at 37°C. A 5-10 ml portion of the blood broth growth is used to inoculate a 2 liter flask containing meat extract nutrient medium. The culture is grown for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. A 14-liter flask containing meat extract nutrient medium is inoculated with the contents of the 2-liter flask and grown for 16 hours at 37°C with the addition of sodium bicarbonate at 2-hour intervals to neutralize the medium. Phenol is then added to the medium to a 1% by volume concentration to kill the bacteria.

An ethanol assay is performed on a sample of the phenolized whole broth to determine the ethanol range of polysaccharides. The range for this rate is from 23 to 35% by volume.

While stirring and at room temperature, 1.79 1 of ethanol is slowly added to 6 1 of the phenolized whole broth, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15-000 r/min in a 10 cm Sharples centrifuge. The solids are discarded.

While stirring, a further 1.44 T ethanol is added long-

as to the supernatant liquid, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged

at 15-000 r/min in a 10 cm Sharples centrifuge. The supernatant liquid is discarded, and the impure polysaccharide solids are suspended in 0.3 1 3% sodium acetate solution using a high shear mixer. The pH is adjusted to 6.5 with glacial acetic acid, and the mixture re-

stirred for 4 hours to ensure dissolution of the polysaccharide.

The mixture is clarified at 15-000 r/min in a 10 cm Sharples centrifuge. Clarification is completed in a K-2 ultracentrifuge at 25,000-30,000 r/min. The solids are discarded.

The pH of the supernatant liquid is adjusted to 7.0 with glacial acetic acid, and 60 mg of MgCl2>0.6 mg of ribonuclease and 0.6 mg of deoxyribonuclease are added. The solution is dissolved with stirring at 37°C for 60 minutes. The pH is adjusted to 8>0 with 50% sodium hydroxide, 12 mg of trypsin is added, and digestion is continued at 37°C for a further 90 minutes. The mixture is cooled to 20-25°C, and the pH is adjusted to 6.5 with glacial acetic acid.

The trapped solution is diafiltered on a "Romicon hollow filter ult rafilt rat ion membrane" until the electrical conductivity of the permeate reaches a constant value. The reservoir is kept at a constant volume with pyrogen-free water. The retentate containing the polysaccharide is drained from the system, and the volume is reset to 1.0 1 with system washer.

The pH of the polysaccharide-containing liquid is adjusted to 6.5 with glacial acetic acid, and an isopropanol assay is performed on a sample of the liquid to determine the optimal isopropanol range for polysaccharide binding. With this rate, the range is from 32-47%. While stirring, 0.14 l of isopropanol is added, and the resulting mixture is aged for 4 hours. The mixture is clarified in a 10 cm Sharples centrifuge at 15,000 r/min. Clarification is completed in a K-2 ultracentrifuge at 25,000-30,000 r/min. The solids are discarded and a further 0.125 1 of isopropanol is added to the supernatant liquid, and the mixture is aged for 4 hours with stirring. The impure polysaccharide precipitate is recovered by centrifugation at 15-000 rpm in a 10 cm Sharples centrifuge, and the supernatant liquid is discarded.

Using a high-shear mixer, the impure polysaccharide is suspended in 0.3 1 sodium acetate solution, and the pH is adjusted to 6.5 with glacial acetic acid. 0.6 1 of isopropanol is added with stirring, and the mixture is stirred for 2 hours and allowed to settle for several hours until a gummy precipitate is separated at the bottom. The entire liquid above is decanted, and the precipitate is triturated in a 'Waring blender at approx. 40 ml of absolute ethanol until a white powder is obtained. The powder is extracted by filtering on a Nutsch

(Whatman No. 2 paper) while washing with 2 x 5 ml absolute ethanol and 2 x 5 ml' acetone. The washed powder is then dried at 686 mm Hg and room temperature for 12-16 hours, whereby approx. 0.72 g of the purified polysaccharide.

Example 7

Pneumococcal polysaccharide type 9

A culture of Streptococcus pneumoniae type 9 is grown on

blood agar for l6 hours at 37°C. The cells are collected and placed in 250 ml of blood broth for 16 hours at 37°C. A 5-10 ml portion of the blood broth growth is used to inoculate a 2 liter flask containing meat extract nutrient medium. The culture is grown for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. A 14-liter flask containing meat extract nutrient medium is inoculated with the contents of the 2-liter flask and grown for 16 hours at 37°C with the addition of sodium bicarbonate at 2-hour intervals to neutralize the medium. Phenol is then added to the medium to a 1% by volume concentration to kill the bacteria.

An ethanol assay is performed on a sample of the phenolized whole broth to determine the ethanol range for polysaccharide precipitation. The range for this rate was from 45 to 60% by volume.

While stirring and at room temperature, 4>91 is added

ethanol slowly to 6 1 of the phenolized whole broth, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15-000 r/min in a 10 cm Sharples centrifuge. The solids are discarded.

C While stirring, a further 4.1 1 ethanol is added slowly

to the supernatant liquid, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15-000 r/min in a 10 cm Sharples centrifuge. The supernatant liquid is discarded, and the impure polysaccharide solids are suspended in 0.6 1 3% sodium acetate solution using a high-shear mixer. The pH is adjusted to 6.5 with glacial acetic acid, and the mixture is stirred for 4 hours to ensure dissolution of the polysaccharide.

An isopropanol test is performed on a sample of the polysaccharide-containing liquid to determine the optimal isopropanol range for polysaccharide sweeping. The range of this rate is from

32 to 47 vol.%.

While stirring, 0.282 1 of isopropanol is added, and the

the resulting mixture is aged for 4 hours with continuous stirring. The mixture is clarified at 15-000 r/min in a 10 cm Sharples centrifuge. Clarification is completed in a K-2 ultracentrifuge at 25,000-30,000 r/min. The solids are discarded. While stirring, a further 0.25 1 of isopropanol is added to the above liquid,

and the resulting mixture is allowed to age for 4 hours with stirring. The impure polysaccharide precipitate is recovered by centrifugation at 15-000 rpm in a 10 cm Sharples sieve, and the supernatant is discarded.

The impure polysaccharide is suspended using a high-shear mixer in 0.6 1 0.9% sodium chloride. The pH is adjusted to 8>0 with 50% sodium hydroxide, 12 mg of trypsin is added, and the solution is digested at 37°C for 90 minutes. The mixture is cooled to 20-25°C, and the pH is adjusted to 6.5 with glacial acetic acid.

The solution is cooled to 0-5°C, 285 g of ammonium sulfate are added, and the mixture is aged at 0-5°C for 4 hours. The precipitate that forms is collected by centrifugation at 15-000 r/min in a 10 cm Sharples centrifuge. Discard the excess liquid.

The precipitate is dissolved in 0.3 1 of pyrogen-free water, and the solution is diafiltered in a "Romicon hollow filter ultrafiltrate ion membrane" until the electrical conductivity of the permeate reaches a constant value. The reservoir is kept at a constant volume with pyrogen-free water. The retentate, containing the polysaccharide, is drained from the system, and the volume is restored to 0.3 1 with system washer. 9 g of sodium acetate are added to the solution of the polysaccharide-containing liquid, and the pH is adjusted to 6.5 with glacial acetic acid.

While stirring, 0.6 1 of isopropanol is added, and the obtained

mixture is stirred for 2 hours and allowed to settle for several hours until a gummy precipitate is separated at the bottom. The entire liquid above is decanted, and the precipitate is triturated in a Waring blender with approx. 40 ml of absolute ethanol until a white powder is obtained. The powder is recovered by filtration on a Nutsch (Whatman No. 2 paper) while washing with 2 x 5 ml of absolute ethanol and 2 x 5 ml of acetone. The washed powder is then dried at 686 mm Hg

and room temperature for 12-16 hours, whereby you get approx. 1.68 g of the purified polysaccharide.

Example 8

Pneumococcal polysaccharide type 10

A culture of Streptococcus pneumoniae type 10 received from the American Type Culture Collection (ATCC No. 63IO), and stored

as a lyophilized culture, suspend in 1 ml of "Difco Heart Infusion Broth" (HIB Broth) and spread 0.2 ml on a rabbit blood agar plate. After 18 hours of incubation at 37°C, the growth on the plate is resuspended in 5 ml of HIB broth, and 1 ml of the suspension is transferred into 100 ml of rabbit blood liquid medium and incubated as a stationary culture for 18 hours at 37°C. After incubation, the culture is examined microscopically and streaked on YED and HIB plates to check purity. The 100 ml culture was stored in a refrigerated

store at 4°C. 10 ml of the culture of the incubated broth was used to inoculate 1 liter of inoculum medium in a 2 liter Erlenmeyer flask. The flask was incubated stationary for 18.5 hours at 37°C. The pH of the fermentation was controlled (6.4-7.0) by adding 12% sodium bicarbonate until an o.d (optical density) of

2.2 had been reached. The 1-liter fermentation requires 80 ml of sodium bicarbonate to reach the above-mentioned o.d. Before inoculation in the next step, a sample is applied to YED and HIB plates to check purity. A sample was also examined microscopically and an agglutination test was performed.

1 liter of the fermentation broth from the previous step

was used to inoculate a 14-liter fermenter containing

9 1 production medium. The batch was incubated at 37°C under gentle agitation (100 r/min) without air flow. The course of the fermentation was followed by determining the o.d., pH and glucose.

After the last sodium hydroxide addition, the fermenter

none continued for another 1 hour and then ended (total time 9 hours). The final o.d. before harvest was 4.24-

The pH was regulated during fermentation (6.2-7.0) using 10% sodium hydroxide. A total of 725 ml of 10% NaOH was used. After the batch was finished, the culture was plated on YED and HIB plates, examined for purity by Gram stain and identified

with regard to the type of the agglutination reaction. The broth became 1 n^Vf n trort WD/^ ri rA o + -i r-\ r~\ ^^th - y- m-* Irr^- h - f q t-» ^ 1 / Q dOZ\ +- - i 1 o r-» c 1 11 + + Vnn -

late ration of 1%. After 2 hours in phenol, the broth was released to recover the product. A sample was taken and applied to HIB-

plate to check viability.

An ethanol assay was performed on a sample of the phenolized whole broth to determine the ethanol range of polysaccharides. The range for this rate was from 48 to 66% by volume. 95% denatured ethanol was added to 10 1 of the broth at a rate of 100 ml/min with constant stirring until the ethanol concentration was 48% by volume. The broth was then clarified by centrifugation at 30,000 r/min in a Sharples centrifuge,

model T-l-P.

To clear the drain, ethanol was added to 66% by volume calculated on the original aqueous system. The precipitate was collected by removing the supernatant with a sieve and by centrifuging the remaining supernatant plus impure polysaccharide in a "Sorvall RC5" at 6,000 r/min (6,089 xg) for 20 minutes at 15°C.

The pellets were resuspended in 1,000 ml of 3% sodium acetate

using a Waring blender at low speed. The pH was adjusted to 6.5.

Isopropanol examination showed that the polysaccharide precipitates

between 38 and 47 volume% isopropanol at room temperature. Isopropanol was therefore added dropwise while stirring on a "thermolyne" stirring plate, middle setting, to a concentration of 38% by volume. The suspension was then clarified by centrifugation in a "Beckman J21" at 14,000 r/min (30,050 xg) at 15°C for 20 minutes. The clear supernatant liquid was diluted with isopropanol to a final concentration of 47% by volume. This led to the separation of a precipitate which was collected by removing the overlying liquid (yellow, brown colour) with a sieve.

The precipitated polysaccharide is resuspended in 1,000 ml of water and the pH is adjusted to 7.0 with concentrated acetic acid. 203 mg of MgCl2

(1 mM), 8 mg of deoxyribonuclease and 8 mg of ribonuclease were added

while stirring. The suspension is incubated in a rotating water bath for 90 minutes at 37°C - After incubation, the pH was set to 8.0

with 1.. N sodium hydroxide, and 49 mg of trypsin were added. The digest was incubated as above.

The pH was adjusted to 7.0, and the digest was transferred into a 29 mm dialysis tube and dialyzed against 14 L of water with one change of water. The dialysis product, 1080 ml, was recovered and brought to 3% sodium acetate. Isopropanol testing indicates that the purified polysaccharide contains between 40 and 52 vol.% isopropanol at room temperature. The sweeping was carried out as in the previous isopropanol sweeping. The polysaccharide adhering to the bottom of the beaker was collected by removing the final supernatant with a sieve. It was triturated in a Waring blender at high speed with 500 ml of absolute ethanol and poured into a medium sintered glass funnel. The polysaccharide was washed with 500 ml of ethanol followed by 250 ml of acetone. Excess solvent was removed by suction, and the polysaccharide dried in vacuum over calcium sulphate. The yield was 7.9 g of pneumococcal polysaccharide type 10.

Example 9

Pn eumococcis k polysaccharide type 12

A culture of Streptococcus pneumoniae type 12 was grown on blood agar for 16 hours at 37°C. The cells were collected and placed in 250 ml of blood broth for 16 hours at 37°C. A 5-10 ml portion of the blood broth growth was used to inoculate a 2 liter flask containing meat extract nutrient medium. The culture was grown for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. A 14 liter flask containing meat extract nutrient medium was inoculated with the contents of the 2 liter flask and cultured for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. Phenol was then added to the medium at 1% vol concentration to kill the bacteria.

An ethanol assay was performed on a sample of the phenolized whole broth to determine the ethanol range of polysaccharides. The range for this rate was from 4-2 to 58% by volume.

While stirring and at room temperature, 4.35 1 of ethanol was slowly added to 6 1 of the phenolized whole broth, and the resulting mixture was aged for 4 hours with continuous stirring. The mixture was then centrifuged at 15-000 rpm in a 10 cm Sharples centrifuge. The solids were discarded.

While stirring, an additional 3.94 1 of ethanol was slowly added

. added to the supernatant liquid, and the resulting mixture became

aged for 4 hours with continuous stirring. The mixture was then centrifuged at 15,000 rpm in a 10 cm Sharples centrifuge.

The supernatant was discarded and the crude polysaccharide solids were suspended in 0.3 1 3% sodium acetate solution using a high shear mixer. The pH was adjusted to 6.5 with glacial acetic acid and stirred for 4 hours to ensure dissolution of the polysaccharide.

An isopropanol study was performed on a sample of the polysaccharide containing liquid to determine the optimal isopropanol range for polysaccharide sweeping. The range for this rate was from 32 to 47% by volume.

While stirring, 0.14l 1 isopropanol was added, and it

the resulting mixture was aged for 4 hours with continuous stirring. The mixture was centrifuged at 15-000 rpm in a >10 cm Sharples centrifuge. Clarification was completed in a K-2 ultracentrifuge at 25,000-30,000 r/min. The solids were discarded. With stirring, an additional 0.125 L of isopropanol was added to the supernatant, and the resulting mixture was allowed to age for 4 hours with stirring. The crude polysaccharide precipitate was recovered by centrifugation at 15-000 rpm in a 10 cm Sharples centrifuge, and the supernatant was discarded.

Using a high shear mixer, the crude polysaccharide was suspended in 0.3 L pH 7.0, 0.05 M phosphate buffer. The pH was readjusted to 7.0 with glacial acetic acid and 60 mg of MgCl 2 , 0.6 mg of ribonuclease and 0.6 mg of deoxyribonuclease were added. The solution was incubated at 37°C for 60 minutes. The pH was adjusted to 8.0 with 50% sodium hydroxide, 12 mg of trypsin was added, and digestion was continued at 37°C for a further 90 minutes. The mixture was cooled to 20-25°C, and the pH was adjusted to 6.5 with glacial acetic acid!

The solution was cooled to 0-5°C, 1449 ammonium sulfate

was added and the mixture was aged at 0-5°C for 4 hours. The precipitate that formed was collected by centrifugation at 15,000 rpm in a 10 cm Sharples centrifuge. The excess liquid was discarded.

The precipitate was dissolved in 0.3 1 pyrogen-free water, and

ning was diafiltered in a "Romicon hollow filter ultra-

filtration membrane" until the electrical conductivity of the permeate reached a constant value. The reservoir was kept at constant volume with pyrogen-free water. The retentate, containing the polysaccharide, was drained from the system, and the volume was again brought to 0.3 L with system washers. 9 g of sodium acetate was added to the solution of the polysaccharide-containing liquid, and the pH was adjusted to 6.5 with glacial acetic acid.

While stirring, 0.6 1 of isopropanol was added, and the resulting mixture was stirred for 2 hours and allowed to settle for several hours until a gummy precipitate had separated at the bottom. The entire supernatant liquid was decanted, and the precipitate was triturated in a Waring blender with approx. /fO ml of absolute ethanol until a white powder was obtained. The powder was recovered by filtration on a Nutsch (Whatman No. 2 paper) washing with 2 x 5 ml absolute ethanol and 2 x 5 ml acetone. The washed powder was then dried at 686 mm Hg and room temperature for 12-16 hours, whereby approx. 1.2 g of the purified polysaccharide.

Example 10

Penumococcal polysaccharide type 14

A culture of Streptococcus pneuraoniae type 14 was grown on blood agar for 16 hours at 37°C. The cells were collected and placed in 250 ml of blood broth for 16 hours at 37°C. A 5-10 ml portion of the blood broth growth was used to inoculate a 2 liter flask containing meat extract nutrient medium. The culture was grown for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. A 14 liter flask containing meat extract nutrient medium was inoculated with the contents of the 2 liter flask and grown for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. Phenol was then added to the medium to a 1 vol% concentration to kill the bacteria.

An ethanol assay was performed on a sample of the phenolized whole broth to determine the ethanol range of polysaccharidin. The range for this rate was from 38 to 53% by volume.

While stirring and at room temperature, 3>71 of ethanol was slowly added to 6 l of the phenolized whole broth, and the resulting mixture was aged for 4 hours with continued stirring. The mixture was then centrifuged at 15-000 rpm in a 10 cm Sharples centrifuge. The solids were discarded.

While stirring, a further 3.1 1 of ethanol was slowly added

was added to the supernatant, and the resulting mixture was aged for 4 hours with continuous stirring. The mixture was then centrifuged at 15-000 rpm in a 10 cm Sharples centrifuge.

The supernatant was discarded, and the crude polysaccharide solids were suspended in 0.5 L pyrogen-free water and stirred for 4 hours to ensure dissolution of the polysaccharide. 12 1 of a 5% sodium acetate solution was added, and the pH was adjusted to

6.5 with glacial acetic acid.

An isopropanol study was performed on a sample of the polysaccharide containing liquid to determine the optimal isopropanol range for polysaccharide sweeping. The range for this rate was from 30 to 42% by volume.

While stirring, 0.25 1 of isopropanol was added, and the

the resulting mixture was aged for 4 hours with continuous stirring. The mixture was centrifuged at 15,000 rpm in a 10 cm Sharples centrifuge. Clarification was completed in a K-2 ultracentrifuge at 25,000-30,000 r/min. The solids were discarded. With stirring, a further 0.18 1 of isopropanol was added to the supernatant liquid from the ultracentrifuge, and the resulting mixture was allowed to age for 4 hours with stirring. The crude polysaccharide precipitate was recovered by centrifugation at 15-000 rpm in a 10 cm Sharples late rif uge and the supernatant was discarded.

The precipitate was dissolved in 0.3 1 of pyrogen-free water, and the solution was diafiltered in a "Romicon hollow filter ultrafilt rate ion membrane" until the electrical conductivity of the permeate reached a constant value. The reservoir was kept at constant volume with pyrogen-free water. The retentate, which contained the polysaccharide, was drained from the system and the volume again brought to 0.3 1 with a system washer. The solution was clarified in a K-2 ultra-centrifuge at 20,000-25,000 r/min.

The clarified solution was cooled to 0-5°C. 30 ml of a solution of 6.6 g of cetavalone in pyrogen-free water was added, whereby a final concentration of 2% was obtained. The mixture was aged for 4 hours at 0.5°C and clarified in the K-2 ultracentrifuge at 20,000-25,000 r/min. 66 g of sodium acetate was added to the supernatant, and the pH was adjusted to 6.5 with glacial acetic acid.

An isopropanol study was performed on a sample of the suspension to determine the optimal isopropanol range for polysaccha rid uniting. With this rate, the range was from 29 to 40% by volume. With stirring, 0.14 L of isopropanol was added, and the resulting mixture was aged for 4 hours. The mixture was clarified in a 10 cm Sharples centrifuge at 15-000 rpm. Clarification was completed in a K-2 ultracentrifuge at 25,000-30,000 r/min. The solids were discarded, and an additional 0.085 L of isopropanol was introduced into the supernatant from the K-2 ultracentrifuge, and the resulting mixture was aged for 4 hours with stirring. The crude polysaccharide precipitate was recovered by centrifugation at 15-000 rpm in a 10 cm Sharples centrifuge, and the supernatant was discarded.

The precipitate was triturated in a Waring blender with approx. 40 ml of absolute ethanol until a white powder was obtained. The powder was recovered by filtration on a Nutsch (Whatman No. 2 paper)

while washing with 2 x 5 ml absolute ethanol and 2 x 5 ml acetone.

The washed powder was then dried at 686 mm Hg and room temperature

trip for 12-16 hours, whereby 1.1 g of the purified polysaccharide was obtained.

Example 11

Pneumococcal polysaccharide type 15

A lyophilized culture of Streptococcus pneuraoniae type 15

was suspended in 1 ml "Difco Heart Infusion Broth" (HIB Broth)

and 0.2 ml was spread on a rabbit blood agar plate. After 18 hours of incubation at 37°C, the growth on the plate was suspended in 5 ral HIB broth, and 1 ml of the suspension was transferred into 100 ml of rabbit blood liquid medium and grown as a stationary culture for 18 hours at 37°C. After incubation, the culture was examined microscopically and streaked

on YED and HIB plates to check purity. 100 ml of the culture was stored in a refrigerator at 4°C.

10 ml of the culture of the incubated broth was used for

to inoculate 1 liter of inoculum medium in a 2-liter Erlenmeyer flask. The flask was incubated stationary for 19 hours at 37°C. pH of fer-

the concentration was regulated (6.4-7)0) by adding 12% sodium bicarbonate up to an o.d. of 2.9 was reached. The 1-liter fermentation required 100 ml of sodium bicarbonate to reach the above-mentioned o.d. Before inoculating the next step, a sample was placed on YED and HIB plates to check the purity. A sample was also examined microscopically and an agglutination test was performed to check the purity and type.

1 liter of the fermentation broth from the previous step

was used to inoculate a 14-liter fermenter containing

9 1 production medium. The batch was incubated at 37°C under gentle agitation (100 r/min) without air flow. The course of the fermentation was monitored by o.d., pH and glucose determinations.

After the last addition of sodium hydroxide, the fermentation was continued for a further 1 hour and then ended (total time 8 hours). The final o.d. before harvest was 4>0. The pH was regulated during the fermentation (6.2-7>0) by using 10% sodium hydroxide. A total of 720 ml of 10% sodium hydroxide was used.

After the batch was finished, the culture was plated on YED-

and HIB plates, examined for purity by Gram stain and identified as to type by the agglutination reaction. Bul-

The young were inactivated by harvesting in pre-liquefied phenol (89%) to a final concentration of 1%. After 2 hours in phenol, the broth was released for extraction of the product. A sample was taken and plated on HIB to check viability.

An ethanol assay was performed on a sample of the phenolized whole broth to determine the ethanol range of polysaccharides. The range for this rate was from 63 to 72 volura%. 95% denatured alcohol was added to 10 1 of the broth at a flow rate of 100 ml/min until the alcohol concentration was 63% by volume. The broth was then centrifuged in a Sharples centrifuge model T-1-P at 30,000 r/min.

95% denatured alcohol was added to the clear effluent

to a final concentration of 72% by volume calculated on the original

equal aqueous volume. The suspension was left overnight at room temperature. As the polysaccharide did not precipitate, sodium acetate was added to a final concentration of 1%. The impure polysaccharide was precipitated and collected by removing the clear supernatant

end liquid with a siphon, The polysaccharide was suspended in 5%-ig nat r iumacet at .

Isopropanol investigation showed that the polysaccharide precipitates between 40 and 50 vol% isopropanol at room temperature. Therefore, isopropanol was added dropwise while stirring on a "thermolyne" magnetic stirrer, middle position, to a concentration of 1+ 0% by volume. The suspension was clarified by centrifugation in a Beckman J21 centrifuge at 14-000 r/min (approx. 30,050 xg) for 20 minutes at 4°C in 250 ml cups. The clear supernatant was brought to 50 vol% isopropanol. The precipitated polysaccharide was collected by removing the supernatant with a sieve.

The precipitate was suspended in 1,000 ml of water. 0.203 g of MgCl2

(1 mM), 8 m9ribonuclease and 7 mg of deoxyribonuclease were added with stirring as in the previous step. The pH was adjusted to

7.0 with glacial acetic acid, and the mixture was incubated for 90 minutes at 37°C in a rotary shaker water bath, setting 2-3, shaker model G76, New Brunswick Scientific. After incubation, the pH was raised to 8.0, 50 mg of trypsin (Worthington) was added with stirring, and the digest was incubated as above for 90 minutes.

The pH was adjusted to 6.5, and the digest was transferred i

16 mm dialysis tube and dialyzed against 14 1 water.

The dialyzed solution (1030 ml) was brought to 5% sodium acetate. Isopropanol examination indicates that the polysaccharide falls between 45 and 52 volume% isopropanol. Fractionation and precipitation were carried out as in the previous isopropanol step. The final precipitated polysaccharide was collected by removing the supernatant with a sieve. The precipitate was scraped into a Waring blender, triturated with 500 ml absolute ethanol and washed in a medium sintered glass funnel. The polysaccharide was further washed with 500 ml of absolute ethanol followed by 250 ml of acetone. Excess of the solvent was removed by suction, and the final product was dried in vacuum over calcium sulfate. The yield was 4.0 g.

Example 12

Pneumococcal polysaccharide type 17

A lyophilized culture of Streptococcus pneumoniae type 17 was suspended in 1 ml of "Difco Heart Infusion Broth" (HIB broth), and 0.2 ml spread on a rabbit blood agar plate. After 18 hours of incubation at 37°C, the growth on the plate was suspended in 5 ml of HIB broth, and 1 ml of the suspension was transferred into 100 ml of rabbit blood liquid medium and incubated as a stationary culture for 18 hours at 37°C. After incubation, the culture was examined microscopically and streaked on YED and HIB plates to check purity. The 100 ml culture was kept in a refrigerator at 4°C 10 ml of the culture of the incubated broth was used to inoculate 1 liter of inoculum medium in a 2-liter Erlenmeyer flask. The flask was incubated stationary for 19 hours at 37°C. The pH of the fermentation liquid was regulated (6.4 - 7.0) by adding 12% sodium bicarbonate until an o.d. of 3.68 was reached. 1 liter of fermentation broth consumes 100 ml of sodium bicarbonate to reach the above O.D. Before inoculating the next step, a sample was applied to YED and HIB plates to check purity. A sample was also examined microscopically, and an agglutinin in ngs sample was carried out to check purity and type. 1 liter of the fermentation liquid from the previous step was used to inoculate a 14 liter fermenter containing 9 liters of production medium. The batch was incubated at 37°C under gentle agitation (100 r/min) without air flow. After the last sodium hydroxide addition, the fermentation was continued for a further 1 hour and then ended (8 hours in total). The final o.d. before harvesting it was 4.4 - pH was regulated during fermentation (6.2-7.0) using 10% sodium hydroxide. A total of 695 ml of 10% sodium hydroxide was used.

After the batch was completed, the culture was plated on YED and HIB plates, examined for purity by Gram stain, and identified as to type by the agglutination reaction. The broth was inactivated by harvesting in 89% pre-liquefied phenol to a final concentration of 1%. After 2 hours in phenol, the broth was released to recover the product. A sample was taken and plated on HIB to check viability.

After ethanol examination, 10 1 of the broth was brought to 50 volume% ethanol (95% denatured ethanol) by adding the alcohol under constant stirring at a flow rate of 100 ml/min. The broth was then centrifuged in a Sharples centrifuge model T-1-P, 30,000 r/min.

To clear the drain, 95% denatured ethanol was added to a final concentration of 57% by volume calculated on the original aqueous volume. The suspension was left overnight at room temperature during which time the precipitate settled.

The impure polysaccharide was collected by removing the supernatant liquid with a sieve, and the precipitate was dissolved in 1,000 ml of 3% sodium acetate.

Isopropanol examination indicated that the polysaccharide precipitates between 35 and 42 vol.% isopropanol at room temperature. Isopropanol was therefore added dropwise while stirring on a magnetic stirrer, to a concentration of 35% by volume. The suspension was cleared by centrifugation in a Beckman J21 at 14,000 r/min (approx. 30,050 xg) for 20 minutes at 4°C. The clear supernatant was brought to 42% isopropanol, and the polysaccharide was collected by removing the supernatant with a sieve.

The precipitate was dissolved in 1.OOO ml of water. The pH was adjusted to 7.0, and 0.2039 MgCl 2 (1 mM), 8 mg of ribonuclease and 8 mg of deoxyribonuclease were added with stirring as in the previous step. The mixture was incubated for 90 minutes at 37°C in a rotating shaking water bath. After incubation, 49 mg of trypsin (Worthington) was added. The digestion was brought to pH 8.0 with

IN NaOH and incubated for 90 minutes as above.

The pH was adjusted to 6.5, and the digest was transferred i

15.9 mm dialysis tube and dialyzed against 14 1 water.

980 ml was recovered and brought to 3% sodium acetate. Iso-propanol examination showed that the polysaccharide precipitates between 40 and 47 volume% isopropanol. Fractionation and precipitation were carried out as in the previous isopropanol step. The final precipitated polysaccharide was collected by removing the supernatant with a sieve. The polysaccharide was scraped into a Waring blender, triturated with 500 ml absolute ethanol and washed onto a medium sintered glass funnel. It is further washed with 500 ml of absolute ethanol followed by 250 ml of acetone. Excess solvent is removed by suction, and the final product is dried in vacuum over CaSO^. Yield: 3.3 g.

Example 13

Pneumococcal polysaccharide type 19

A culture of Streptococcus pneuraoniae type 19 was grown on blood agar for 16 hours at 37°C. The cells were collected and placed in 250 ml of blood broth for 16 hours at 37°C. A 5-10 ml portion of the blood broth growth was used to inoculate a 2 liter flask containing meat text rakt nutrient medium. The culture is grown for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. A 14-liter flask containing meat extract nutrient medium was inoculated with the contents of the 2-liter flask and grown for 16 hours at 37°C

while adding sodium bicarbonate at 2 hour intervals to neutralize the medium. Phenol was then added to the medium to a 1 vol% concentration to kill the bacteria.

An ethanol assay was performed on a sample of the phenolized whole broth to determine the ethanol range of polysaccharides. The range for this rate was from 48 to 62% by volume.

While stirring and at room temperature, 5.54 1 of ethanol was slowly added to 6 1 of the phenolized whole broth, and the resulting mixture was aged for 4 hours with continuous stirring.

Eng. The mixture was then centrifuged at 15-000 rpm in a 10 cm Sharples centrifuge. The solids were discarded.

While stirring, an additional 4525 1 of ethanol was slowly added to the supernatant liquid, and the resulting mixture was aged for 4 hours with continuous stirring. The mixture was then centrifuged at 15-000 rpm in a 10 cm Sharples centrifuge.

The supernatant was discarded and the crude polysaccharide solids were suspended in 0.6 1 3% sodium acetate solution using a high shear mixer. The pH was adjusted to 6.5 with glacial acetic acid and stirred for 4 hours to ensure dissolution of the polysaccharide.

An isopropanol study was performed on a sample of the polysaccharide containing liquid to determine the optimal isopropanol range for polysaccharide sweeping. The range for this rate was from 35 to 45% by volume.

While stirring, 0.323 1 of isopropanol was added, and the resulting mixture was aged for 4 hours with continuous stirring.

Eng. The mixture was centrifuged at 15,000 rpm in a 10 cm Sharples centrifuge. Clarification was completed in a K-2 ultracentrifuge at 25,000-30,000 r/min. The solids were discarded. With stirring, a further 0.168 L of isopropanol was added to the supernatant liquid, and the resulting mixture was allowed to age for 4 hours with stirring. The crude polysaccharide fraction 11 was recovered by centrifugation at 15,000 rpm in a 10 cm Sharples centrifuge, and the supernatant was discarded.

The crude polysaccharide was suspended using a high shear mixer in 0.3 L pH 7.0, 0.05 M phosphate buffer. The pH was again adjusted to 7.0 with glacial acetic acid and 60 mg MgCl 2 , 0.3 m 9 ribonuclease and 0.3 m 9 deoxyribonuclease were added. The solution was dissolved under stirring at 37°C for 60 minutes. The pH was adjusted to 8.0 with 50% NaOH, 0.6 mg of trypsin was added, and the digestion was continued at 37°C for 90 minutes. The mixture was cooled to 20-25°C, and the pH was adjusted to 6.5 with glacial acetic acid.

The solution was cooled to 0-5°C, 180 g of ammonium sulfate

was added and the mixture was aged at 0-5°C for 4 hours. The precipitate that formed was collected by centrifugation at 15,000 rpm in a 10 cm Sharples centrifuge. The excess liquid was discarded.

The precipitate was dissolved in 0.3 1 pyrogen-free water, and the solution was diafiltered in a "Romicon hollow filter ult rafiltration membrane" until the electrical conductivity of the permeate reached a constant value. The reservoir was kept at constant volume with pyrogen-free water. The retentate, which contained the polysaccharide, was drained from the system and the volume again brought to 0.3 1

with system washer.

9 g of sodium acetate was added to the solution of the polysaccharide-containing liquid, and the pH was adjusted to 6.5 with glacial acetic acid.

With stirring, 0.6 l of isopropanol was added, and it formed

mixture was stirred for 2 hours and allowed to settle for several hours until a gummy precipitate separated at the bottom. The entire supernatant liquid was decanted, and the precipitate was triturated in a Waring blender with approx. 40 ml of absolute ethanol until a white

powder was obtained. The powder was recovered by filtration on a

nutsch (Whatman No. 2 paper) while washing with 2 x 5 ml absolute ethanol and 2 x 5 ml acetone. The washed powder was then dried at 686 mm Hg and room temperature for 12-16 hours, whereby approx. 5.49 of the purified polysaccharide.

Example 14

Pneumococcal polysaccharide type 20

A culture of Streptococcus pneumoniae type 20 (ATCC 6320) is suspended in 1 ml of "Difco Heart Infusion Broth" (HIB Broth) and 0.2 ml is spread on a rabbit blood agar plate. After 18 hours of incubation at 37°C, the growth on the plate is suspended in 5 ml of HIB broth, and 1 ml of the suspension is transferred into 100 ml of rabbit blood liquid medium and incubated as a stationary culture for 18 hours at 37°C. After incubation, the culture is examined microscopically and streaked on YED and HIB plates to check purity. The 100 ml culture is saved

in a refrigerator.at 4°C.

10 ml of the incubated culture is used to inoculate

1 liter of inoculum medium in a 2-liter Erlenmeyer flask. The flask is incubated stationary for 18 hours at 37°C. The pH of the fermentation liquid is regulated (6.4~7.0) by adding 12% bicarbonate up to an o.d. of 2.8 is reached. 1 liter of fermentation liquid consumes 85 ml of sodium bicarbonate to reach the above-mentioned etc. Before inoculating the next step, a sample is streaked onto YED and HIB plates to check purity. A sample is also examined microscopically, and an agglutination test is performed to check purity and type. 1 liter of fermentation broth from the previous step is used to inoculate a 14 liter fermenter containing 9 liters of production medium. The batch is incubated at 37°C under gentle agitation (100 r/min) without air flow. The progress of the fermentation is monitored by o.d., pH and glucose determinations.

After the last addition of sodium hydroxide, the fermentation is continued for a further 2 hours and then terminated (total time 10.5 hours). The final o.d. before harvest is 3.1. The pH is regulated during fermentation (6.2-7.0) using 10% NaOH. A total of 700 ml of 10% NaOH is used.

After the batch is finished, the culture is streaked onto YED and HIB plates, examined for purity by Gram staining, and identified with respect to type by the agglutination reaction. The broth is inactivated by harvesting in 89% pre-liquefied phenol to a final concentration of 1% by volume. After 2 hours in phenol, the broth is released, and the product is recovered. A sample is taken and pressed

HIB to check viability.

After ethanol examination, 95% denatured alcohol is added

hol to 10 1 of the broth to bring the alcohol content on

53% by volume. Alcohol is added with constant stirring at a flow rate of 100 ml/min. The broth is then centrifuged in a Sharples centrifuge model T-1-P, at 30,000 r/min.

To clear the drain, a further 95% denatured alcohol is added to a final concentration of 65% by volume calculated on the original aqueous system. The insoluble material (which falls immediately upon addition of the alcohol) is collected by removing the supernatant liquid with a sieve and by centrifugation in a "Sorvall RC5" at 10,000 r/min (16.3OO xg) in 500 ml cups at 23° C for approx. 15 minutes.

The pellets are suspended in 1,000 ml of 3% sodium acetate.

Isopropanol examination shows that the polysaccharide falls

between 44 and 52 volume% isopropanol at room temperature. Isopropanol is therefore added dropwise while stirring on a "thermolyne" stirring plate, middle setting, to a 44% volume level. The suspension is then centrifuged in a "Sorvall RC5" in 250 ml cups at 14,000 r/min for 15 minutes at 23°C (approx. 31,700 xg). The clear supernatant liquid is diluted with isopropanol to a concentration of 52% by volume. This leads to the secretion of a gum which is collected by removing the excess liquid with a siphon.

The rubber is suspended in 1,000 ml pyrogen-free water. The pH is adjusted to 7.0, 203 mg MgCl2 (1 mM), 4 mg ribonuclease and 6 mg deoxyribonuclease are added while stirring as in the previous step. This is incubated in a shaking water bath for 1 hour at 37°C. After incubation, the pH is adjusted to 8.0 with NaOH, and 30 mg of trypsin is added while stirring as in the previous step. The mixture is incubated for 90 minutes as above.

The pH is adjusted to 7.0, and the digest is transferred into a 15.9 mm dialysis tube and dialyzed against 14 1 of water with one change of water.

The dialyzed solution is diluted with 2 volumes of 95% denatured alcohol. Addition of 300 ml of an alcoholic electrolyte (1 volume of 20% sodium acetate, pH 6.5, plus 2 volumes of 95% denatured alcohol) leads to precipitation of the polysaccharide. The polysaccharide is collected by centrifugation on a Beckman J21 in 500 ml cups at 10,000 r/min (17,680 xg) for 15 minutes at 23°C. The pellets are transferred to a Waring blender and triturated with 500 ml of 95% denatured ethanol. The solid particles are washed onto a medium sintered glass funnel and washed again with 500 ml of 95% denatured ethanol and 250 ml of acetone. After removal of excess solvent by suction, the polysaccharide is dried in vacuum over CaSO^. The dividend is 4.39-

Example 15

Pn eumococcal polysaccharide type 23

A culture of Streptococcus pneumoniae type 23 is grown on blood agar for 16 hours at 37°C. The cells are collected and placed in 250 ml of blood broth for 16 hours at 37°C. A 5-10 ml portion of the blood broth growth is used to inoculate a 2 liter flask containing meat extract nutrient medium. The culture is grown for 16 hours at 37°C while sodium bicarbonate is added

2 hour intervals to neutralize the medium. A 14-liter flask containing meat extract nutrient medium is inoculated with the contents of the 2-liter flask and cultured for 16 hours at 37°C

while adding sodium bicarbonate at 2 hour intervals to neutralize the medium. Phenol is then added to the medium to a 1% by volume concentration to kill the bacteria.

An ethanol assay is performed on the sample of the phenolized whole broth to determine the ethanol range of polysaccharide precipitation. The range for this rate is from 48 to 62% by volume.

While stirring and at room temperature, 5.54 1 of ethanol is added slowly to 6 1 of the phenolized whole broth, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15-000 rpm in a 10 cm Sharples centrifuge. The solids are discarded.

While stirring, a further 4.25 1 ethanol is added lang-

as to the liquid above, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is centrifuged

then at 15,000 r/min in a 10 cm Sharples centrifuge. The supernatant liquid is discarded, and the impure polysaccharide solids are suspended in 0.6 1 3% sodium acetate solution using a high-shear mixer. The pH is adjusted to 6.5 with glacial acetic acid and stirred for 4 hours to ensure dissolution of the polysaccharide.

An isopropanol test is performed on a sample of the polysaccharide-containing liquid to determine the optimal isopropanol range for polysaccharide sweeping. The range for this rate is from 33 to 48% by volume.

While stirring, 0.295 1 of isopropanol is added, and the

the resulting mixture is aged for 4 hours with continuous stirring. The mixture is clarified at 15-000 r/min in a 10 cm Sharples centrifuge. Clarification is completed in a K-2 ultracentrifuge at 25,000 - 30,000 r/min. The solids are discarded. While stirring, a further 0.259 1 of isopropanol is added to the liquid above, and the resulting mixture is allowed to age for 4 hours while stirring.

The impure polysaccharide precipitate is recovered by centrifugation at 15,000 r/min in a 10 cm Sharples centrifuge, and the supernatant liquid is discarded.

The impure polysaccharide is suspended using a high shear mixer in 0.6 L pH 7.0, 0.05 M phosphate buffer. The pH is again adjusted to 7.0 with glacial acetic acid and 60 mg of MgClg, 0.6 mg of ribonuclease and 0.6 mg of deoxyribonuclease are added. The solution is dissolved by stirring at 37°C for 60 minutes. The pH is adjusted to 8.0 with 50% NaOH, 18 mg of trypsin is added, and the digestion is continued at 37°C for a further 90 minutes. The mixture is cooled to 20-25°C, and the pH is adjusted to 6.5 with glacial acetic acid.

The solution is cooled to 0-5°C, 5.1 g of NaCl and 240 g of ammonium sulfate are added, and the mixture is aged at 0-5°C for 4 hours. The precipitate that forms is collected by centrifugation at 15-000 r/min in a 10 cm Sharples centrifuge. Discard the excess liquid.

The precipitate is dissolved in 0.3 1 of pyrogen-free water, and the solution is diafiltered in a "Romicon hollow filter ult rafiltrat ion membrane" until the electrical conductivity of the permeate reaches a constant value. The reservoir is kept at a constant volume with pyrogen-free water. The retentate, containing the polysaccharide, is drained from the system and the volume is restored to 0.3 L with

system washer.

9 g of sodium acetate are added to the solution of the polysaccharide-containing liquid, and the pH is adjusted to 6.5 with glacial acetic acid. While stirring, 0.6 1 isopropanol is added, and the resulting mixture is stirred for 2 hours and allowed to settle to the bottom for several hours until a gummy precipitate is separated at the bottom. The entire liquid above is decanted, and the precipitate is triturated in a Waring blender with approx. 40 ml of absolute ethanol until a white powder is obtained. The powder is recovered by filtration on a slide (Whatman No. 2 paper) while washing with 2 x 5 ml of absolute ethanol and 2 x 5 ml of acetone. The washed powder is then dried at 686 mm Hg and room temperature for 12-16 hours, whereby approx. 1.92 g of the purified polysaccharide.

Example 16

Pneumococcal polysaccharide type 25

A culture of Streptococcus pneuraoniae type 25 is grown on blood agar for 16 hours at 37°C. The cells are collected and placed in 250 ml of blood broth for 16 hours at 37°C. A 5-10 ml portion of the blood broth growth is used to inoculate a 2 liter flask containing meat extract nutrient medium. The culture is grown for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. A 14 liter flask containing meat extract nutrient medium is inoculated with the contents of the 2 liter flask and cultured for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. Phenol is then added to the medium to a 1% by volume concentration to kill the bacteria.

An ethanol assay is performed on a sample of the phenolized whole broth to determine the ethanol range for polysaccharide precipitation. The range for this rate was from 52 to 65% by volume.

While stirring and at room temperature, 6.5 1 of ethanol is added slowly to 6 1 of the phenolized whole broth, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15,000 r/min in a 10 cm Sharples centrifuge. The solids are discarded.

While stirring, a further 4.6" 1 of ethanol is added slowly to the supernatant liquid, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15,000 r/min in a 10 cm Sharples centrifuge. The supernatant liquid is discarded, and the - impure polysaccharide solids are suspended in 0.6 1 3% sodium acetate solution, the pH is adjusted to 6.5 with glacial acetic acid, and the mixture is stirred for 4 hours to ensure dissolution of the polysaccharide.

An isopropanol assay is performed on a sample of the polysaccharide containing liquid to determine the optimal isopropanol range for polysaccharide sweeping. The range for this rate was from 38 to 52% by volume.

While stirring, 0.37 1 of isopropanol is added, and the resulting mixture is aged for 4 hours with constant stirring. The mixture is clarified at 15,000 r/min in a 10 cm Sharples centrifuge. Clear-

none is completed in a K-2 ultracentrifuge at 25-000-30,000 r/min. The solids are discarded. While stirring, a further 0.3 1 of isopropanol is added to the liquid above, and the resulting mixture is aged for 4 hours while stirring. The impure polysaccharide precipitate is recovered by centrifugation at 15-000 rpm in a 10 cm Sharples centrifuge, and the supernatant liquid is discarded.

The impure polysaccharide is suspended in 0.3 1 pyrogen-free

water. The pH is adjusted to 7.0 with glacial acetic acid, and 0.12 g of MgCl 2 , 3.0 mg of ribonuclease and 3.0 mg of deoxyribonuclease are added. The solution is dissolved with stirring at 37°C for 60 minutes. The pH is adjusted to 8.0 with 50% NaOH, 0.5 g of trypsin is added, and digestion is continued at 37°C for a further 90 minutes. The mixture of

cooled to 20-25°C, and pH adjusted to 6.5 with glacial acetic acid.

The trapped solution is diafiltered in a "Romicon hollow filter ult rafilt rat ion membrane" until the electric wire

ability of the permeate reaches a constant value. The reservoir is kept at a constant volume with pyrogen-free water. The retentate, which contains the polysaccharide, is diverted from the system, and the volume is brought back to 0.3 1 with a system washer.

The pH of the polysaccharide-containing liquid is adjusted to 6.5 with glacial acetic acid, and an isopropanol test is performed on a sample of the liquid to determine the optimum isopropanol concentration for polysaccharide formation. With this rate, the range was from 38 to 47% by volume. While stirring, 0.18 1 of isopropanol is added, and the mixture obtained was aged for 4 hours. The mixture is clarified in a 10 cm Sharples centrifuge at 15-000 r/min. The clearance is completed in one

K-2 ultracentrifuge at 25,000-30,000 r/min. The solids are discarded, and a further 0.082 1 of isopropanol is introduced into the supernatant liquid from the K-2 centrifuge, and the resulting mixture is aged for 4 hours with stirring. The impure polysaccharide

precipitate is recovered by centrifugation at 15-000 rpm in a 10 cm Sharples late rif week, and the supernatant liquid is discarded.

The precipitate is triturated in a Waring blender with approx. 40 ml of absolute ethanol until a white powder is obtained. The powder out-

is obtained by filtration on a nutsch (Whatman No. 2 paper) while washing with 2 x 5 ml of absolute ethanol and 2 x 5 ml of acetone. The washed powder is then dried at 686 mm Hg and room temperature for 12-16 hours, whereby approx. 2.5 g of the purified polysaccharide.

Example 17

Pneumococcal polysaccharide type 51

A culture of Streptococcus pneumonia type 51 is grown on

blood agar for l6 hours at 37°C. The cells are collected and placed in 250 ml of blood broth for 16 hours at 37°C. A 5-10 ml portion of the blood broth growth is used to inoculate a 2 liter flask containing meat extract nutrient medium. The culture is grown for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. A 14 liter flask containing meat extract nutrient medium is inoculated with the contents of the 2 liter flask and cultured for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. Phenol is then added to the medium to a 1% by volume concentration to kill the bacteria.

An ethanol test is carried out on a sample of the phenolic

tease whole broth to determine the ethanol range of polysaccharide precipitation. The range for this rate was from 50 to 70% by volume.

While stirring and at room temperature, add 6 1

ethanol slowly to 6 L of the phenolized whole broth, and the resulting mixture was aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15-000 r/min in a 10 cm Sharples centrifuge. The solids are discarded.

While stirring, a further 8 1 of ethanol are added slowly

to the supernatant liquid, and the resulting mixture is aged for 4 hours with continuous stirring. The mixture is then centrifuged at 15-000 r/min in a 10 cm Sharples centrifuge. The supernatant liquid is discarded, and the impure polysaccharide solids are suspended in 0.6 1 3% sodium acetate solution using a high-shear mixer. The pH is adjusted to 6.5 with glacial acetic acid, and mix-

none is stirred for 4 hours to ensure dissolution of the polysaccharide.

An isopropanol assay is performed on a sample of the polysaccharide containing liquid to determine the optimum isopropanol range for polysaccharide precipitation. The range for this rate is from 42 to 5.7% by volume.

While stirring, 0.434 1 isopropanol is added slowly, and

the resulting mixture is aged for 4 hours with continuous stirring. The mixture is clarified at 15-000 r/min in a 10 cm Sharples centrifuge. Clarification is completed in a K-2 ultracentrifuge at 25,000-30,000 r/min. The solids are discarded. While stirring, a further 0.361 1 of isopropanol is slowly added to the

liquid, and the resulting mixture is allowed to age for 4 hours with stirring. The impure polysaccharide precipitate is recovered by centrifugation at 15-000 r/min in a 10 cm Sharples centrifuge.

joint, and the excess liquid is discarded.

The impure polysaccharide is suspended using a high shear mixer in 0.3 L pH 7.0, 0.05 M phosphate buffer. The pH is again adjusted to 7.0 with glacial acetic acid and 60 mg of MgCl2, 0.6 mg of ribonuclease and 0.6 mg of deoxyribonuclease are added. The solution is dissolved with stirring at 37°C for 60 minutes. The pH is adjusted to 8.0 with 50% NaOH, 18 mg of trypsin is added, and digestion is continued at

37°C for a further 90 minutes. The mixture is cooled to 20-25°C,

and the pH is adjusted to 6.5 with glacial acetic acid.

The trapped solution is diafiltered in a "Romicon hollow filter ult rafilt rat ion membrane" until the electric wire

ability of the permeate reaches a constant value. The reservoir is kept at a constant volume with pyrogen-free water. The retentate, which contains the polysaccharide, is drained from the system, and the volume is restored to 0.3 1 with system washer. 9 g of sodium acetate are added to the solution of the polysaccharide-containing liquid, and the pH is adjusted to 6.5 with glacial acetic acid.

While stirring, 0.6 1 of isopropanol is added, and the obtained

mixture is stirred for 15 minutes and allowed to settle for several hours until a gummy precipitate is separated at the bottom. The entire liquid above is decanted, and the precipitate is triturated in a Waring blender with approx. 40 ml of absolute ethanol until a white

powder is available. The powder is recovered by filtration on a Nutsch (Whatman No. 2 paper) while washing with 2 x 5 ml of absolute ethanol and 2 x 5 ml of acetone. The washed powder is then dried at 686 mm Hg and room temperature for 12-16 hours, whereby approx. 3j339

purified polysaccharide.

Example 18

Pneumococcal polysaccharide type 56

A culture of Streptococcus pneuraoniae type 56 is grown on blood agar for 16 hours at 37°C. The cells are collected and placed in 250 ml of blood broth for 16 hours at 37°C. A 5-10 ml portion of the blood broth growth is used to inoculate a 2 liter flask containing meat extract nutrient medium. The culture is grown for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. A 14 liter flask containing meat extract nutrient medium is inoculated with the contents of the 2 liter flask and cultured for 16 hours at 37°C adding sodium bicarbonate at 2 hour intervals to neutralize the medium. Phenol is then added to the medium to a 1% by volume concentration to kill the bacteria.

An ethanol assay was performed on a sample of the phenolized whole broth to determine the ethanol range of polysaccharides. The range for this rate was from 52 to 67% by volume.

While stirring and at room temperature, 6.5 1 of ethanol was added slowly to 6 1 of the phenolized whole broth, and the resulting mixture was aged for 4 hours with continuous stirring.

The mixture was then centrifuged at 15,000 r/min in a 10 cm

Sharples centrifuge. The solids were discarded.

While stirring, an additional 5>68 L of ethanol was added slowly to the supernatant liquid, and the resulting mixture was aged for 4 hours with continuous stirring. The mixture was then centrifuged at 15-000 rpm in a 10 cm Sharples centrifuge.

The supernatant was discarded and the crude polysaccharide solids were suspended in 0.6 1 3% sodium acetate solution using a high shear mixer. The pH was adjusted to 6.5 with glacial acetic acid and stirred for 4 hours to ensure dissolution of the polysaccharide.

An isopropanol study was performed on a sample of the polysaccharide containing liquid to determine the optimal isopropanol range for polysaccharide sweeping. The range for this rate was from 38 to 50% by volume.

While stirring, 0.368 1 of isopropanol was added, and the

the resulting mixture was aged for 4 hours with continuous stirring. The mixture was centrifuged at 15-000 rpm in a 10 cm Sharples centrifuge. Clarification was completed in a K-2 ultracentrifuge at 25,000-30,000 rpm. The solids were discarded. With stirring, a further 0.232 L of isopropanol was added to the supernatant liquid, and the resulting mixture was allowed to age for 4 hours with stirring. The crude polysaccharide precipitate was recovered by centrifugation at 15-000 r/min in a 10 cm Sharples centrifuge,

and the excess liquid was discarded.

The crude polysaccharide was suspended using a high shear mixer in 0.3 L pH 7.0, 0.05 M phosphate buffer. The pH was again adjusted to 7.0 with glacial acetic acid and 60 mg MgCl 2 , 0.6 mg ribonuclease and 0.6 mg deoxyribonuclease were added. The resulting solution was dissolved under stirring at 37°C for 60 minutes. The pH was adjusted to 8.0 with 50% NaOH, 18 mg of trypsin was added, and the digestion was continued at 37°C for a further 90 minutes. The mixture was cooled to 20-25°C, and the pH was adjusted to 6.5 with glacial acetic acid.

The solution was cooled to 0-5°C, 2.55 g of NaCl and 120 g of ammonium sulfate were added, and the mixture was aged at 0-5°C for 4 hours. The precipitate that formed was collected by centrifugation at 20,000-25,000 r/min in the K-2 ultracentrifuge. The excess liquid was discarded.

The precipitate was dissolved in 0.3 1 pyrogen-free water, and the solution was diafiltered in a "Romicon hollow filter ultrafiltration membrane" until the electrical conductivity of the permeate reached a constant value. The reservoir was kept at constant volume with pyrogen-free water. The retentate containing the polysaccharide was decanted from the system, and the volume was again brought to 0.3 L with system wash. 9 g of sodium acetate was added to the solution of the polysaccharide-containing liquid, and the pH was adjusted to 6.5 with glacial acetic acid.

While stirring, 0.6 1 of isopropanol was added, and the resulting mixture was stirred for 2 hours and allowed to settle for several hours until a gummy precipitate had separated at the bottom. The entire supernatant liquid was decanted, and the precipitate was triturated in a Waring blender with approx. L\ Q ml of absolute ethanol until a white powder was obtained. The powder was recovered by filtration on a Nutsch (Whatman No. 2 paper) washing with 2 x 5 ml absolute ethanol and 2 x 5 ml acetone. The washed powder was then dried at 686 mm Hg and room temperature for 12-16 hours, whereby approx. 2.04 g of the purified polysaccharide.

Example 19

Pneumococcal polysaccharide type 57

A culture of Streptococcus pneuraoniae type 57 was suspended

in 1 ml "Difco Heart Infusion Broth" (HIB broth), and 0.2 ml spread on a rabbit blood agar plate. After 18 hours of incubation at 37°C

the growth on the plate was suspended in 5 ml of HIB broth, and 1 ml of the suspension was transferred into 100 ml of rabbit blood liquid medium and incubated as a stationary culture for 18 hours at 37°C. After incubation, the culture was examined microscopically and streaked on YED-

and HIB plates to check purity. The 100 ral culture was stored in a refrigerator at 4°C.

10 ml of the incubated culture was used to inoculate

1 liter of inoculum medium in a 2-liter Erlenmeyer flask. The flask was incubated stationary for 18 hours at 37°C. The pH of the fermentation medium was regulated (6.4-7.0) by adding 12% sodium bicarbonate up to an o.d. of 2.0 had been reached. The 1-liter fermentation consumed 100 ml of sodium bicarbonate in obtaining the above, etc. Before inoculating the next step, a sample was streaked on YED and HIB plates to check purity. A sample was also examined microscopically, and an agglutination sample

was performed to check purity and type.

1 liter of fermentation broth from the previous step was used to inoculate a 14 liter fermenter containing 9 liters of production medium. The batch was incubated at 37°C under gentle agitation (100 rpm) without air flow. The course of the fermentation was monitored by o.d.-, pH- and glucose-determining Is er. After the last sodium hydroxide addition, the fermentation was continued for a further 1 hour and then ended (total time 10.5 hours).

The final o.d. before harvest was 5.0. The pH was regulated during the fermentation (6.2-7.0) using 10% igNaOH. A total of 655 ml of 10% NaOH was used.

When the batch was finished, the culture was plated on YED and HIB plates, examined for purity by Gram stain and identified

with regard to the type of the agglutination reaction. The broth was inactivated by harvesting in 89% pre-liquefied phenol to a final concentration of 1%. After 2 hours in phenol, the broth was released to recover the product. A sample was taken and applied to the HIB to check viability.

After ethanol examination, 10 1 of the broth was added

50 volume% ethanol by adding 95% denatured alcohol at a flow rate of 100 ml/min under constant stirring. The broth was then clarified by centrifugation in the Sharples centrifuge

at 40,000 r/min.

A further 95% denatured alcohol was added to the clear effluent to 65% by volume calculated on the original aqueous system. The precipitate was collected by removing most of the upper

supernatant with a sieve and by centrifuging the remaining supernatant plus the impure polysaccharide in a "Sorvall RC5" at 6,000 r/min (6,089 xg) for 20 minutes at 5°C.

The pellets were suspended in 1,000 ml of 3% sodium acetate using a Waring blender at low speed. The pH was adjusted to 6.5.

Isopropanol examination showed that the polysaccharide falls

between 30 and 42 volume% isopropanol at room temperature. Isopropanol was therefore added dropwise while stirring on a "themolyne" stirring plate, in the middle, to a concentration

of 30% by volume. The suspension was then clarified by centrifugation in a Beckman J21 at 14,000 r/min (30,050 xg) at 15°C for 20 min-

otter. The clear supernatant liquid was diluted with isopropanol to a final concentration of 42%. This leads to the separation of a precipitate which is collected by centrifugation on a Beckman J21 at 6,000r/min for 15 minutes at 5°C (6,370 xg).

The precipitated polysaccharide is suspended in 1,000 ml of water, and

The pH is adjusted to 7.0 with glacial acetic acid. 203 m 9 MgCl 2 (1 mM), 10 mg deoxyribonuclease and 15 mg ribonuclease are added with stirring. The mixture is incubated in a rotating water bath for 80 minutes at

37°C After incubation, the pH is adjusted to 8>0 with 1 N NaOH, and 30 mg of trypsin is added. The suspension is incubated as above.

The pH is adjusted to 7) 0, and the digest is transferred into a 28.6 mm dialysis tube and dialyzed against 14 1 of water with a change of water.

About. 1,000 ml is extracted and brought to 3% sodium acetate. Isopropanol examination indicates that the purified polysaccharide

add between 40 and 50 volume% isopropanol at room temperature. The precipitation is carried out as in the previous isopropanol step. The polysaccharide that adheres to the bottom of the beaker is collected by removing the final excess liquid with a sieve. It is triturated in a Waring blender at high speed with 500 ml of absolute ethanol and poured into a medium sintered glass funnel. The polysaccharide is washed with 500 ml of ethanol followed by 250 ml of acetone. Excess solvent is removed by suction, and the polysaccharide is dried in vacuum over CaSO^. The yield is 3>7g-

Example 20

Pneumococcal polysaccharide type 72

A culture of Streptococcus pneumoniae type 72 is suspended in

1 ml "Difco Heart Infusion Broth" (HIB Broth), and 0.2 ml spread on a rabbit blood agar plate. After 18 hours of incubation at 37°C, the growth on the plate is suspended in 5 ml of HIB broth, and 1 ml of the suspension is transferred into 100 ml of rabbit blood liquid medium and incubated as a stationary culture for 18 hours at 37°C. After incubation, the culture is examined. microscopically and streaked on YED and HIB plaques to check purity. The 100 ml culture is saved

in a refrigerator at 4°C.

10 ml of the incubated culture is used to inoculate

1 liter of inoculum medium in a 2-liter Erlenmeyer flask. The flask is incubated stationary for 19 hours at 37°C. The fermentation medium's pH is regulated (6.4-7.0) by adding 12% sodium bicarbonate up to an o.d. of 1.24 is reached. 1 liter of the fermentation consumes 55 ml of sodium bicarbonate to reach the above-mentioned o.d. Before inoculating the next step, a sample is streaked onto YED and HIB plates to check purity. A sample is also examined microscopically, and an agglutination test is performed to check purity and type.

1 liter of the fermentation broth from the previous step

was used to inoculate a 14-liter fermenter containing

9 liters of production medium. The batch was incubated at 37°C under gentle agitation (100 r/min) without air flow. The course of the fermentation was followed by o.d., pH and glucose determinations. When the o.d. determinations were just over a 2-hour period, the fermenter

none terminated (total incubation time 10 hours). The final o.d. before harvesting was 3.68 - pH was regulated during fermentation (6.2-7.0) using 10% NaOH. A total of 600 ml of 10% NaOH was used.

After the batch was finished, the culture was streaked on YED-

and HIB plates, examined for purity by Gram stain and identified as to type by the agglutination reaction. The broth was inactivated by harvesting in 89% pre-liquefied phenol to a final concentration of 1%. After 2 hours in phenol, the broth was released to recover the product. A sample was taken and applied to HIB-

plates to check viability.

After the ethanol test, the broth was brought to 42% ethanol by volume by adding 95% denatured ethanol. The alcohol was added slowly with constant stirring at a flow rate of 100 ml/min. The broth was then centrifuged in a Sharples centrifuge, model T-1-P, at 30,000 r/min.

To clear the drain, 95% denatured ethanol was added

to a final concentration of 57% by volume calculated on the original aqueous system. The insoluble material (which precipitates immediately upon addition of the alcohol) was collected by removing the excess liquid with a sieve, and by late rif ugation in a Beck

man J21 at 9,000 r/min (approx. 14-000 xg) at 23°C for 20 minutes.

The pellets were suspended in 1,000 ml of 3% sodium acetate,

pH 6.5.

Isopropanol examination showed that the polysaccharide precipitates

between 30 and 45 vol% isopropanol at room temperature. Isopropanol was therefore added dropwise with stirring to a 30% volume level. The suspension was then centrifuged in a Beckman J21 in 250 ml cups at 10,000 r/min at 23°C

(15-380xg). The clear supernatant liquid was diluted with isopropanol to a concentration of 45% by volume. This led to the secretion of a semi-liquid gum which was collected by

remove the excess liquid with a sieve.

The gum was suspended in 1,000 ml of pyrogen-free water. pH

was set to 7.0. 203 mg of MgCl 2 (1 mM), 4 mg of ribonuclease and 6 mg of deoxyribonuclease were added with stirring. The mixture was incubated in a rotating water bath for 1 hour at 37°C. After incubation, the pH was adjusted to 8.0 with NaOH, and 30 mg of trypsin was added with stirring. The mixture was incubated for 90 minutes as above.

The pH was adjusted to 6.5, and the digest was transferred i

15.9 mm dialysis tubing and dialyzed against 14 1 water with one change of water.

The dialyzed solution (950 ml) was diluted with

2 volumes of 95% denatured ethanol. Addition of 150 ml of an alcoholic electrolyte (1 volume of 20% sodium acetate, pH 6.5,

plus 2 volumes of 95% denatured ethanol) led to precipitation of the polysaccharide. The polysaccharide was collected by removing the supernatant with a sieve, and the "mat" was transferred to a Waring blender and triturated with 500 ml of 95% denatured ethanol.

The solid particles were washed onto a medium sintered glass

funnel and washed again with 500 ml of 95% denatured ethanol and 250 ml of acetone. After removal of excess solvent by suction, the polysaccharide was dried in vacuum over CaSO 4 . The yield was 7.3 g.

Example 21

Pneumococcal polysaccharide type 73

A lyophilized culture of Streptococcus pneuraoniae type 73 was suspended in 1 ml of "Difco Heart Infusion Broth" (HIB Broth) and 0.2 ml was spread on a rabbit blood agar plate. After 18 hours of incubation at 37°C, the growth on the plate was suspended in 5 ml of HIB broth and 1 ml of the suspension was transferred into 100 ml of rabbit blood liquid medium and incubated as a stationary culture for 18 hours at 37°C. After incubation, the culture was examined microscopically and streaked on YED and HIB plates to check purity. The 100 ml culture was stored in a refrigerator at 4°C.

10 ml of the incubation culture was used to inoculate

1 liter of inoculum medium in a 2-liter Erlenmeyer flask. The flask was incubated stationary for 16 hours at 37°C. The pH of the fermentation was regulated (6.4-7.0) by adding 12% sodium bicarbonate up to an o.d. of 1.4 was reached. The 1-liter fermentation consumed 120 ml of sodium bicarbonate to reach the above-mentioned o.d.

Before inoculation in the next step, a sample was streaked onto YED and HIB plates to check purity. A sample was also examined microscopically, and an agglutination test was performed to check purity and type. 1 liter of the fermentation broth from the previous step was used to inoculate a 14 liter fermenter containing 9 liters of production medium. The batch was incubated at 37°C under gentle agitation (100 r/min) without air flow. The course of the fermentation was monitored by o.d., pH and glucose determinations. When the o.d. determinations were just over a 2-hour period, the fermentation was terminated (17 hours total incubation). The final o.d. before harvest was 4.48- PH was regulated during the fermentation (6.2-7.0) using 10%-igNaOH. A total of 665 ml of 10% NaOH was used.

After the batch was completed, the culture was streaked onto YED and HIB plates, examined for purity by Gram stain, and identified as to type by the agglutination reaction. The broth was inactivated by harvesting in 89% pre-liquefied phenol to a final concentration of 1%. After 2 hours in phenol, the broth was released to recover the product. A sample was taken and applied to HIB plates to check viability.

After ethanol examination, the broth was brought to /+0 vol.% alcohol by adding 95% denatured ethanol. The alcohol was added slowly with constant stirring at a flow rate of 100 ml/min. The broth was then centrifuged in a Sharples centrifuge, model T-1-P, at 30,000 r/min.

To clear the drain, 95% denatured ethanol was added

to a final concentration of 6o volume% calculated on the original

equally aqueous system. The suspension was left overnight at room temperature during which time the precipitate settled.

The insoluble material (impure polysaccharide) was collected by

to remove the supernatant clear liquid with a sieve and centrifuge in a Beckman J21 at 5,000 r/min (approx. 4-000 xg) for 10 minutes at 23°C in 500 ml cups.

The pelletized material was suspended in 1,000 ml of 3% sodium acetate buffer, pH 6.5, with stirring on a thermolyne stirring plate, medium setting.

Isopropanol investigation indicated that the polysaccharide precipitates between 20 and 42 vol% isopropanol at room temperature. Isopropanol was therefore added dropwise with stirring to a concentration of 20% by volume, and the suspension was centrifuged in a Beckman J21 at 10,000 r/min (15-380 xg) in 250 ml cups at 23°C. The clear supernatant liquid was further diluted with isopropanol to a concentration of 42% by volume. This led to the secretion of a "semi-liquid gum" which was collected by

remove the supernatant liquid with a sieve and centrifugation in a Beckman J21 at 6,000 r/min (approx. 5-520 xg) in 250 ml cups at

23°C

The pellet from the previous step was dissolved in 1,400 ml

1% sodium acetate buffer. pH was adjusted to 7.0, 284 mg MgCl 2

(1 mM), 1 mg ribonuclease and 1 mg deoxyribonuclease were added

while stirring as in the previous step. The mixture was incubated in a rotating shaking water bath for 1 hour at 37°C. After incubation, the pH was adjusted to 8.0 with 1 M NaOH, and 30 mg of trypsin was added with stirring. The mixture was incubated as above for 90 minutes.

The pH was adjusted to 6.5, and the digest was transferred into 15.9 mm dialysis tubing and dialyzed against 14 L of water (changed three times).

The dialyzed solution (1,680 ml) was diluted with 2 volumes of 95% denatured ethanol. The addition of 150 ml of an alcoholic electrolyte (1 volume of 20% sodium acetate plus

2 volumes of 95% denatured ethanol) led to immediate flocculation of the polysaccharide. The polysaccharide was collected by centrifugation in a Beckman J21, 250 ml cups, for 15 minutes at 10,000 rpm (15-380 xg) at 23°C. The pellet was transferred into a Waring blender and was triturated with 500 ml of 95% denatured ethanol. The solid particles were washed onto a medium sintered glass funnel with 500 ml of 95% denatured ethanol followed by 250 ml of acetone. After removal of excess solvent by suction, the polysaccharide was dried in vacuum over CaSO 4 . The yield was 7.9 g-

Example 22

A polyvalent vaccine was prepared by adding, under aseptic conditions at room temperature, the following amounts of capsular polysaccharide to 3-160 ml of pyrogen-free saline (0.85%). The polysaccharides were added individually at 15 second intervals with continuous stirring in a mixer. After the last polysaccharide was added, stirring was continued for a further 2-3 minutes.

The mixture was then sterile filtered and packed in ampoules, 6 ml (10 doses) per ampoule.

Claims (6)

1. Method for the production of a purified capsular polysaccharide of Streptococcus pneuraoniae, characterized in that a predetermined amount of a water-miscible alcohol is added to a liquid medium containing the polysaccharide and thereby traps impurities, the impurities are separated from the liquid medium, a predetermined amount of a water-miscible alcohol is added to the liquid medium to precipitate the polysaccharide, the polysaccharide is suspended in a liquid medium, and proteins and nucleic acids are removed from the polysaccharide. 2. Method according to claim 1, characterized in that a fermentation broth is used as the liquid medium. 3- Method according to claim 1 or 2, characterized in that an alcohol with 2 or 3 carbon atoms is used as water-miscible alcohol. 4' Method according to claims 1-3, characterized in that proteins and nucleic acids are removed by treatment with trypsin and nucleases. 5- Method according to claims 1-3, characterized in that proteins and nucleic acids are removed by treatment with a cationic surfactant. 6. Method according to claim 5, characterized in that cetrimonium bromide is used as a cationic surfactant. 7- Method according to claims 4-6, characterized in that protein fragments and nucleic acid fragments are removed by diafiltration.